Compositions and methods for the diagnosis and treatment of immune disorders

ABSTRACT

The present invention relates to methods and compositions for the treatment and diagnosis of immune disorders, especially T helper lymphocyte-related disorders. In particular, the invention describes a gene known in the art, alternatively, as ST2, T1 and Fit-1, and referred to herein as the 103 gene. The 103 gene is disclosed herein to be differentially expressed in TH2 cells and not in TH1 cells. Further, the 103 gene product is demonstrated herein to be an important modulator of TH2 and TH2-like immune response both in vitro and in vivo. Thus, the 103 gene, its gene products and antibodies that specifically bind thereto can be used diagnostically or as targets for therapeutic intervention in the treatment of a variety of immune disorders. 
     In this regard, the invention provides methods for the identification and therapeutic use of compounds for treatments of immune disorders, especially TH cell subpopulation-related disorders and including TH2 and TH2-like disorders (i.e., disorders associated with a TH2 or TH2-like mediated immune response) such as atopic conditions (e.g., allergy and asthma). Additionally, methods are provided for the diagnostic evaluation and prognosis of TH cell subpopulation related disorders, for the identification of subjects exhibiting a predisposition to such conditions, for monitoring patients undergoing clinical evaluation for the treatment of such disorders and for monitoring the efficacy of compounds used in clinical trials.

This application claims priority under 35 U.S.C. §119 (e) to U.S.provisional application Ser. No. 60/155,862, filed on Sep. 24, 1999,which is incorporated herein, by reference, in its entirety.

1. FIELD OF THE INVENTION

The present invention relates to methods and compositions for thetreatment and diagnosis of immune disorders, especially Tlymphocyte-related disorders, including, but not limited to, chronicinflammatory disease and disorders (e.g., Crohn's disease, reactivearthritis, and Lyme disease), insulin-dependent diabetes, organ specificautoimmunity (including, e.g., multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease), contact dermatitis, psoriasis, graftrejection, graft versus host disease, sarcoidosis, atopic conditions(e.g., asthma and allergy including, but not limited to, allergicrhinitis and gastrointestinal allergies such as food allergies),eosinophilia, conjunctivitis, glomerular nephritis, systemic lupuserythematosus, scleroderma, certain pathogen susceptibilities such ashelminthic (e.g., leishmaniasis) and certain viral infections (includingHIV and bacterial infections such as tuberculosis and lepromatousleprosy).

In particular, the methods and compositions of the present inventionrelate to detection and/or modulation of expression and/or activity of agene product referred to herein as the 103 gene, as well as to detectionand/or modulation of expression and/or activity of gene products encodedby the 103 gene (i.e., a “103 gene product”).

2. BACKGROUND OF THE INVENTION

The majority of mature T lymphocytes can be divided into two distinctphenotypes: CD8⁺ cytotoxic T lymphocytes (CTLs), which display the CD8marker on their cell surface, and CD4⁺ helper T lymphocytes (T helper orTH cells), which display the CD4 marker on their cell surface. Thissubdivision is also associated with functional differences between thetwo cell types CTLs are, in general, involved in cell-mediated, orcellular, immune responses, and are activated by intracellular pathogenssuch as, for example, microbes and viruses. In particular, foreignantigens (e.g., viral antigens) are synthesized within infected cellsand presented on the surfaces of such cells in association with class Imajor histocompatibility complex (MHC) molecules. CTL precursors displayT cell receptors that recognize these antigens, triggering activation,maturation and proliferation of the precursor CTLs and resulting in CTLclones capable of destroying the cells exhibiting the antigensrecognized as foreign.

T helper (TH) cells are involved in both humoral (i.e., antibody) andcell-mediated forms of immune response. With respect to the involvementof TH cells in humoral, or antibody, immune response, extracellularantigens are endocytosed by antigen presenting cells (APCs), processedand presented, preferentially in association with class II MHCmolecules, to CD4⁺ class II MHC-restricted TH cells. These TH cells inturn activate B lymphocytes, resulting in antibody production. Withrespect to the role of TH cells in cell-mediated forms of immuneresponse, some agents, such as mycobacteria which cause tuberculosis andleprosy, are engulfed by macrophages and processed in vacuolescontaining proteolytic enzymes and other toxic substances. While thesemacrophage components are capable of killing and digesting mostmicrobes, agents such as mycobacteria survive and multiply. However, theagents' antigens are processed by the macrophages and presented inassociation with class II MHC molecules to CD4⁺ class II MHC-restrictedTH cells. These TH cells, in turn, become stimulated to secreteinterferon-γ (IFN-γ) which activates macrophages. Such activationresults in an increased bacteriocidal ability.

TH cells have been further categorized into two distinct subpopulations,termed TH1 and TH2 cell subpopulations. These two subpopulations of THcells have been categorized on the basis of their restricted cytokineprofiles and different functions. For example, TH1 cells are known toproduce IL-2, tumor necrosis factor β (TNF-β) and IFN-γ. TH2 cells areknown to produce interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin10 (IL-10) and interleukin 13 (IL-13). The different subpopulations arederived from a common precursor, or “naive” TH cell population (referredto as THP), and acquire their set pattern of cytokine production duringa process referred to as “commitment.”

Genetic and environmental factors acting at the level of antigenpresentation influence the commitment of a common naive T cell precursorto TH1 or TH2 differentiation. For example, the conditions of antigenstimulation (including both the nature and amount of antigen involved),the type of antigen-presenting cells and the type of hormone andcytokine molecules present all seem to represent determinants of thepattern of TH1 versus TH2 differentiation from a common naive T helpercell precursor. In particular, the decisive role appears to belong tothe particular cytokines present in the cells environment. For example,IL-4, which is produced by TH2 and TH2-like cells, also appears to be animportant factor in the commitment of naive THP cells to the TH2subtype. Further, once TH1 and TH2 subpopulations are expanded, the twocell types tend to negatively regulate one another through the actionsof cytokines unique to each subpopulation. For example, IFN-γ, which isproduced by TH1 cells, negatively regulates TH2 cells, whileTH2-produced IL-10 negatively regulates TH1 cells. Moreover, cytokinesproduced by TH1 and TH2 antagonize the effector functions of one another(Mosmann, T. R. and Moore, 1991, Immunol. Today 12:49). Although a fullaccounting of the exact factors important in driving TH1 and/or TH2differentiation are, as yet, largely unknown, certain transcriptionfactors activated in response to a given cytokine have been shown to beimportant in TH1 and/or TH2 differentiation. For example, the activationof signal transducer and activator of transcription (STAT)-6 by IL-4 hasbeen shown to be important in TH2 differentiation, and the activation ofSTAT-4 has been shown to be important in TH1 differentiation (e.g.,Romagnani, S., 1997, Immunology Today 18:263-266; Ray, A. and Cohn, L.,1999, The Journal of Clinical Investigation 104(8):985-993).

Although the TH1 and TH2 subtypes were originally identified in murinesystems (see, for example, Mosmann, T. R. and Coffman, R. L., 1989, Ann.Rev. Immunol. 7:145), the existence of TH1-like and TH2-likesubpopulations has also been established in humans (see, e.g., DelPrete, A. F. et al., 1991, J. Cline. Invest. 88:346; Wiernenga, E. A. etal., 1990, J. Imm. 144:4651; Yamamura, M. et al., 1991, Science 254:277;Robinson, D. et al., 1993, J. Allergy Clin. Imm. 92:313; Anderson, G. P.and Coyle, A. J., 1994, Trends in Pharmacological Sciences 15(9):324-32;Romagnani, S., 1997, Immunology Today 18:263-266). Human TH1-like andTH2-like cells have similar cytokine profiles to the TH1 and TH2 cellsoriginally identified in murine systems, and preferentially expressactivation markers (e.g., CD30 and LAG-1). CD30, a member of the tumornecrosis factor (TNF) receptor family, is primarily expressed byTH2-like cells, and lymphocyte activation gene 3 (LAG-3) ispreferentially expressed by TH1-like cells.

TH cells having characteristics (e.g., cytokine production profiles) ofboth TH1 and TH2 cell subpopulations have been designated TH0 cells(see, e.g., Firestein, G. S. et al., 1989, J. Imm. 143:518). CD8⁺ Tcytotoxic (Tc)-cell subpopulations have also been identified based onthe cytokines they produce. In general, activated CD8⁺ CTLs exhibit aTH1-like cytokine profile, but under some conditions CD8⁺ CTLs exhibit aTH2-like cytokine profile (Seder, R. A. et al., 1995, J. Exp. Med.181:5-7; Manetti, R. et al., 1994, J. Exp. Med. 180:2407-2411; Maggi, E.et al., 1994, J. Exp. Med. 180:489-495). As noted above, TH1 and TH2cell subpopulations appear to have great relevance to immune responseagainst infectious agents such as viruses and intracellular parasites.

TH1-like and TH2-like cells appear to function as part of differenteffector functions of the immune system (see, e.g., Mosmann andCoffmann, supra). For example, TH1-like cells direct the development ofcell-mediated immunity, triggering phagocyte mediated host defenses, andare associated with delayed hypersensitivity. Accordingly, infectionswith intracellular microbes tend to induce TH1-type responses. TH2-likecells drive humoral immune responses, which are associated with, forexample, defenses against certain helminthic parasites and are involvedin antibody and allergic responses.

Failure to control or resolve an infectious process often results notfrom an insufficient immune response but, rather, from an inappropriateresponse. Such inappropriate immune responses underlie a variety ofdistinct immunological disorders including, for example, mastocytosis(e.g., cutaneous mastocytosis and systemic mastocytosis), interstitialcystitis (IC), and atopic conditions (e.g., IgE-mediated allergicconditions) such as asthma, allergy (in eluding allergic rhinitis),dermatitis (including psoriasis), systemic lupus erythematosus,scleroderma, pathogen susceptibilities, chronic inflammatory disease,organ-specific autoimmunity, graft rejection and graft versus hostdisease. For example, nonhealing forms of human and murine leishmaniasisresult from strong but counterproductive TH2-like-dominated immuneresponses. Lepromatous leprosy also appears to feature a prevalent butinappropriate, TH2-like response.

Atopic conditions, such as asthma and allergy, are also examples ofdisorders that arise because of a TH2-like response to allergen (see,e.g., Holgate, S. T., 1997, Lancet 350 (suppl. II):5-9; Ray, A. andCohn, L, supra; Oettgen, H. C. and Geha, R. S., 1999, The Journal ofClinical Investigation 104(7):829-835). In particular, such disordersare characterized by the development of IgE antibodies to foreignproteins. IgE antibodies are produced by B cells stimulated with IL-4, acytokine produced by TH2 and TH2-like cells. Moreover, TH2-like cytokineprofiles have been observed, not only in TH cells isolated from patientssuffering from asthma and/or allergy, but also in mast cells and CD8⁺CTLs isolated from such patients (Anderson and Coyle, supra). Further,animal studies have demonstrated that TH2-like cells play an importantrole in the induction of inflammation and the chronic pathologicalchanges associated with asthma. For example, the constitutive expressionof TH2 cytokines (e.g., IL-4 and IL-5) in mice has been shown to inducean asthma-like syndrome (Ray, A. and Cohn, L, supra).

A bias towards a TH2-like response has also been suggested to contributeto the loss of control of the immune system over HIV infection. Inparticular, a drop in the ratio of TH1-like cells to other TH cellsubpopulations has been suggested to play a critical role in theprogression toward disease symptoms. Further, it has been noted that, atleast in vitro, TH2-like clones appear to be more efficient supportersof HIV viral replication than TH1-like clones (Romagnani, S., supra).

Further, while TH1-mediated inflammatory responses to many pathogenicmicroorganisms are beneficial, such responses to self antigens areusually deleterious. It has been suggested that the preferentialactivation of TH1-like responses is central to the pathogenesis of suchhuman inflammatory autoimmune diseases as multiple sclerosis andinsulin-dependent diabetes. For example, TH1-type cytokines predominatein the cerebrospinal fluid of patients with multiple sclerosis,pancreases of insulin-dependent diabetes patients, thyroid glands ofHashimoto's thyroiditis, and gut of Crohn's disease patients, suggestingthat such patients mount a TH1-like, not a TH2-like, response to theantigen(s) involved in the etiopathogenesis of such disorders.

A primary goal, for both diagnostic and therapeutic reasons, therefore,would be the ability to identify, isolate and/or target members of aparticular TH cell subpopulation. As such, the identification of geneswhich are differentially expressed within and/or among TH cellsubpopulations is desirable. To date, investigations have focused on theexpression of a limited number of specific known cytokines and cytokinereceptors in the TH cell population. Cytokines, however, exert effectson cell types in addition to specific TH cell subpopulations, i.e.,exhibit a variety of pleiotropic effects. It would be beneficial,therefore, to identify reliable markers (e.g., gene sequences) of THcell subpopulations whose effects are TH cell subpopulation specific,e.g., which, unlike secreted cytokines, are TH cell subpopulationspecific.

Discussion or citation of a patent, patent publication or otherreference herein shall not be construed as an admission that suchpatent, patent publication or citation is prior art to the presentinvention.

3. SUMMARY OF THE INVENTION

The present invention relates to methods and compositions for thediagnosis of immune disorders and for the treatment (e.g., theamelioration or modulation of symptoms associated with) of immunedisorders, especially T helper (TH) cell and TH cell-like relateddisorders such as the TH cell and TH cell-like related disordersdescribed herein below. The invention relates, in particular, to novelmethods and compositions which use a gene referred to herein as the 103gene or a modulator thereof. The gene is also known, alternatively, asT1, ST2 or Fit-1.

The invention is based, in part, on the discovery of a novel nucleotidesequence depicted in FIG. 21 (SEQ ID NO:24) which encodes a previouslyunknown human 103 gene product, referred to herein as Athdc120c9 (FIG.21; SEQ ID NO:25). The invention is also based, in part, on thediscovery that the 103 gene is expressed, in vivo, in a tightlycontrolled TH2 or TH2-like specific manner, and that the 103 geneproduct is an important molecule in signaling TH2-mediated immuneresponses. In particular, the 103 gene is expressed in a specificsubpopulation of T helper cells (i.e., in TH2 or TH2-like cells and notin TH1 or TH1-like cells). For example, results are presented hereinwhich demonstrate that the 103 gene product plays a critical role as asignaling molecule required for the differentiation and function of TH2and TH2-like cells. In particular, the data presented hereinbelow showthat blockage of 103 gene product signaling suppresses both thedifferentiation and activation of TH2 but not TH1 cell subpopulations.Data are also presented showing that the 103 gene product is a criticalregulatory molecule for TH2-mediated immune responses in vivo. Inparticular, results obtained using animal models for allergy and forasthma are presented herein indicating that the 103 gene productprovides a critical signal to TH2-mediated responses in these disordersand that blockage of this signal ameliorates symptoms associated withthe disorders. For example, the results presented herein in Section 6.4demonstrate successful amelioration of asthma symptoms by administrationof either an anti-103 antibody (i.e., an antibody that specificallybinds to a 103 gene product) or a fusion protein comprising anextracellular or secreted domain of a 103 gene product.

Accordingly, compounds such as natural ligands, derivatives of naturalligands and antibodies that specifically bind to the 103 gene product,can be utilized to modulate (e.g., reduce or increase) the number of TH2and/or TH2-like cells present in a population or system. For example,TH2 or TH2-like cells can be physically separated away from other cellsin a population. Alternatively, the specific destruction of TH2 and/orTH2-like can be targeted. Further, proliferation of TH2 and/or TH2-likecells can be modulated (e.g., induced, increased, inhibited or reduced).Additionally, compounds such as 103 gene sequences or 103 gene productscan be utilized to modulate the level of TH2 or TH2-like cell activityand/or to cause modulation in the level of TH2 cell cytokine production(e.g., such methods can bring about a reduction in the level ofproduction of cytokines, such as IL-4, IL-5, IL-10 and IL-13, that areassociated with TH2 cell subpopulations and/or with TH2 cellsubpopulation activity). For example, IL-4 produced by the TH2 andTH2-like cell subpopulations stimulates B cells which, in turn, produceIgE-type antibodies. Thus conditions that involve an inappropriate IgEimmune response, including but not limited to the symptoms whichaccompany atopic conditions such as allergy and/or asthma, can betreated and/or ameliorated by reducing IL-4 levels, e.g., by using themethods of the present invention to reduce TH2 cell activity.

Given its status as both a TH2 and TH2-like cell subpopulation specificmarker and a critical regulatory molecule, the 103 gene, its geneproducts, and modulators thereof can be used in a variety of novelmethods and compositions described herein to diagnose and/or modulateimmune system disorders, particularly disorders that are known to beassociated with a TH2 or TH2-like cell subpopulation. The 103 gene andits gene products can also be used in a variety of methods andcompositions, which are also described herein, to identify andcharacterize compounds, including, for example, small molecules, thatare useful for prognosis, diagnosis, monitoring, rational drug designand/or therapeutic intervention of immune system disorders. Further,molecules, such as certain monoclonal antibodies, that recognize andspecifically bind to a ligand binding domain of a 103 gene product caninhibit this binding interaction. Thus, the invention also provides forcompounds that inhibit or modulate ligand binding of a 103 gene product.Such compounds can also be used in the methods and compositions of thepresent invention to modulate 103 gene product activity and therebymodulate immune system disorders, including disorders that are known orbelieved to be associated with a TH2 or TH2-like cell subpopulation.

In addition, the 103 gene is also expressed in mast cells. Thus, theinvention also relates to methods and compositions that can also beutilized to modulate other cell populations, such as mast cells, thatspecifically express the 103 gene. In particular, the number of mastcells present and/or the amount of mast cell activity or mast cellcytokine production (e.g., from the degranulation of mast cells) canalso be modulated using the methods and compositions described herein.Thus conditions, including atopic conditions such as asthma and allergy,mastocytosis (e.g., cutaneous mastocytosis and systemic mastocytosis),and interstitial cystitis (IC) that involve or are mediated by mast cellactivity (often in addition to TH2 or TH2-like activity) can be treatedby using the methods and compositions of the invention to target mastcells and/or mast cell activity as well as (or instead of) TH2 cellsand/or TH2 cell activity.

Thus, the present invention relates to methods for the prognostic anddiagnostic evaluation of various TH cell subpopulation-relateddisorders, and for the identification of subjects who are predisposed tosuch disorders. Furthermore, the invention provides methods forevaluating and monitoring the efficacy treatments and therapies forvarious immune disorders, such as for the evaluation of drugs for immunedisorders and for monitoring the progress of patients involved inclinical trials for the treatment of immune disorders.

The invention also relates to methods and compositions that can beutilized in the amelioration of symptoms stemming from such immunedisorders (e.g., from such TH cell subpopulation disorders) and formodulating TH or TH-like cell responsiveness such as, for example,responsiveness to an antigen. For example, such methods can compriseadministering an effective amount of a composition to an individualsexhibiting TH cell subpopulation-related disorders or tendencies so thatone or more symptoms of such disorders or tendencies are modulatedand/or thereby ameliorated. Additionally, the treatment methods providedby the present invention may result in the stimulation or depletion ofone or more of the TH cell subpopulations. “Stimulation,” as the term isused herein, can refer to: (a) an effective increase in the number ofcells belonging to a TH cell subpopulation via, for example, theproliferation of such TH cell subpopulation cells; or (b) an increase inthe activity of cells belonging to a TH cell subpopulation, as would beevidenced, for example, by a per cell increase in the expression of theTH cell subpopulation specific cytokine pattern. “Depletion,” as theterm is used herein, can refer to: (a) an effective reduction in thenumber of cells belonging to a TH cell subpopulation via, for example, areduction in the proliferation of such TH cell subpopulation cells; or(b) a decrease in the activity of cells belonging to a TH cellsubpopulation, as would be evidenced, for example, by a per celldecrease in the expression of the TH cell subpopulation-specificcytokine pattern.

Among the compositions that can be utilized as part of such methods are103 gene sequences; polypeptides comprising 103 gene product amino acidsequences, and antibodies directed against 103 gene products. Inaddition, such compositions can include compositions, such as smallmolecule compositions, that modulate 103 gene expression, and/or 103gene product activity, and can, for example, include compoundsidentified by the screening methods described herein.

The 103 genes or gene sequences used in the methods and compositions ofthe present invention encompasses: (a) at least one of the nucleotidesequences and/or fragments thereof that are depicted herein FIGS. 1, 3A,4A, 5A, 6A, 7A and 8 (SEQ ID NOS:1-5, 10 and 12); (b) any nucleotidesequence or fragment thereof that encodes the amino acid sequenceencoded by one of the nucleotide sequences that are depicted in FIGS. 1,3A, 4A, 5A, 6A, 7A and 8 (SEQ ID NOS:1-5, 10 and 12); (c) any nucleotidesequence that hybridizes to the complement of one of the codingnucleotide sequences depicted herein in FIGS. 1, 3A, 4A, 5A, 6A, 7A and8 (SEQ ID NOS:1-5, 10 and 12) under stringent conditions, e.g.,hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate(SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDSat about 50-65° C., or hybridization to filter-bound DNA in 0.5 M sodiumpyrophate/7% SDS at about 65° C. followed by one or more washes in0.2×SSC/1% SDS at about 42-55° C., or under other stringenthybridization conditions which are known to those of skill in the art(see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocolsin Molecular Biology, Vol. I, Green Publishing Associates, Inc. and JohnWiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3); (d) anynucleotide sequence that hybridizes to the complement of one of thecoding nucleotide sequences depicted herein in FIGS. 1, 3A, 4A, 5A, 6A,7A and 8 (SEQ ID NOS:1-5, 10 and 12) under highly stringent conditions,e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45°C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.,or hybridization to filter-bound DNA in 0.5 M sodium pyrophosphate/7%SDS at about 65° C. followed by one or more washes in 0.2×SSC/1% SDS atabout 68° C., or under other stringent hybridization conditions whichare known to those of skill in the art (see, for example, Ausubel, F. M.et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I,Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New Yorkat pages 6.3.1-6.3.6 and 2.10.3), including such other hybridizationconditions as those described herein; and (e) the complement of any ofthe 103 genes or gene sequences recited in (a)-(d) above.

The TH cell subpopulation-related disorders include, for example, TH1 orTH1-like related disorders (i.e., disorders that are associated with aTH1 or TH1-like mediated immune response). Examples of such disordersinclude chronic inflammatory disease and disorders (e.g., Crohn'sdisease, reactive arthritis and Lyme disease), insulin-dependentdiabetes, organ specific autoimmunity (including multiple sclerosis,Hashimoto's thyroiditis and Grave's disease), contact dermatitis,psoriasis, graft rejection, graft versus host disease and sarcoidosis.The TH cell subpopulation-related disorders further include, forexample, TH2 or TH2-like related disorders (i.e., disorders that areassociated with a TH2 or TH2-like mediated immune response). Examples ofsuch disorders include atopic conditions such as asthma and allergy(including, e.g., allergic rhinitis, gastrointestinal allergies and foodallergies), eosinophilia, conjunctivitis, glomerular nephritis, systemiclupus erythematosus and scleroderma. Other exemplary TH2 and/or TH2-likerelated disorders include certain pathogen susceptibilities such ashelminthic (e.g., leishmaniasis), certain viral infections (including,for example, HIV infection) and bacterial infections (including, forexample, tuberculosis and lepromatous leprosy).

The methods and compositions described herein can also be utilized inthe prognostic and diagnostic evaluation of disorders involving otherimmune cells, including CD8+ cytotoxic T lymphocytes (“CTL's”), thatexhibit or are capable of exhibiting TH-like cell subpopulation geneexpression patterns and/or activities. The methods and compositionsdescribed herein can still further be utilized in the amelioration ofsymptoms stemming from disorders involving such immune cells, especiallysuch CD8+ CTL's, which exhibit TH-like cell subpopulation geneexpression patterns and/or activity.

The present invention also relates to methods for the identification ofcompounds which modulate the expression of genes or the activity (e.g.,level) of gene products involved in TH cell subpopulation-relateddisorders and processes relevant to the differentiation, maintenanceand/or effector function of the subpopulations. For example, presentedherein are methods for identifying compounds that affect the level ofexpression of the 103 gene and/or activity of the 103 gene product.Among such methods are, for example, methods for identifying compoundswhich bind to a 103 gene product.

The present invention encompasses a monoclonal antibody produced by thehybridoma clone M15 3F7.3 (ATCC™ No. PTA-593), the hybridoma clone M152O3.1 (ATCC™ No. PTA-591), the hybridoma clone M15 10F7.1 (ATCC™ No.PTA-592), the hybridoma clone M15 1B4.1 (ATCC™ No. PTA-588), thehybridoma clone M15 9F11.1 (ATCC™ No. PTA-590), the hybridoma clone M155A16.1 (ATCC™ No. PTA-587), or an antigen binding fragment thereof. Anantigen binding fragment of a monoclonal antibody of the inventionrefers to a fragment of the antibody that binds to a 103 gene productsuch as a Fab fragment and an F(ab′)₂ fragment. The present inventionfurther encompasses an isolated antibody that competes with themonoclonal antibody produced by hybridoma clone M15 3F7.3, M15 2O3.1,M15 10F7.1, M15 1B4.1, M15 9F11.1 or M15 5A16.1 for epitope binding. Theisolated antibody can be, e.g., a monoclonal antibody, a single chainantibody, a human antibody or a humanized antibody.

The following terms, as they are used in herein, shall have thedefinitions provided hereinbelow.

The term “aberrant expression,” as used herein to describe theexpression of a 103 gene product, refers to the overexpression orunderexpression of a 103 gene product relative to the level ofexpression of a 103 gene product by cells obtained from a healthysubject or a subject without an immune disorder state, and/or to ahigher or lower level of 103 gene product or transcript in a tissuesample or body fluid obtained from a healthy subject or a subjectwithout an immune disorder state. In particular, a 103 gene product isaberrantly expressed if the level of expression of a 103 gene product ishigher or lower by at least 2 fold, at least 5 fold, at least 10 fold,at least 15 fold, at least 25 fold, or at least 50 fold relative to thelevel of expression of the 103 gene product by cells obtained from ahealthy subject or a subject without an immune disorder state, and/orrelative to the level of expression of the 103 gene product in a tissuesample or body fluid obtained from a healthy subject or a subjectwithout an immune disorder state.

The term “TH cell subpopulation,” as used herein, refers to a populationof TH cells exhibiting a gene expression pattern (e.g., a discretepattern of cytokines and/or receptor or other cell surface molecules)and activity which are distinct from the expression pattern and activityof other TH cells. Such TH cell subpopulations can include, but are notlimited to, TH0, TH1 and TH2 cell subpopulations which will, for clarityand example and not by way of limitation, be frequently used herein asrepresentative TH cell subpopulations. In particular and as noted above(Section 2), although TH cell subpopulations such as TH1 and TH2 cellsubpopulations were originally discovered in murine systems, theexistence of similar TH cell subpopulations (i.e., TH1-“like” andTH2-“like” cell subpopulations) has also been established in otheranimals, including other mammals such as humans. Thus, it is understoodthat the particular TH cell subpopulations referred to herein (e.g.,TH0, TH1 and TH2 cell subpopulations) refer not only to the CD4+ TH cellsubpopulations originally identified in murine systems, but also toequivalent or similar (e.g., functionally equivalent) CD4+ TH cellsubpopulations in other animals, including other mammals such as humans.

The term “TH-like cell subpopulation” (e.g., “TH1-like” or “TH2-like”),therefore, as used herein, can refer, not only to a population of CD4+TH cells having the properties described, above, for a TH cellsubpopulation, but also refers to CD4− cells, including CD8+ CTL's,which exhibit TH-like cytokine expression patterns.

“Differential expression,” as the term is used herein, is understood torefer to both quantitative as well as qualitative differences intemporal and/or cellular expression patterns, e.g., of a gene or genes.

“Negative modulation”, as used herein, refers to a reduction in thelevel and/or activity of target gene product relative to the leveland/or activity of the target gene product in the absence of themodulatory treatment. Alternatively, the term, as used herein, refers toa reduction in the number and/or activity of cells belonging to the THcell subpopulation relative to the number and/or activity of the TH cellsubpopulation in the absence of the modulatory treatment.

“Positive modulation”, as used herein, refers to an increase in thelevel and/or activity of target gene product relative to the leveland/or activity of the gene product in the absence of the modulatorytreatment. Alternatively, the term, as used herein, refers to anincrease in the number and/or activity of cells belonging to the TH cellsubpopulation, relative to the number and/or activity of the TH cellsubpopulation in the absence of the modulatory treatment.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. In one embodiment, a nucleic acidmolecule is cDNA and not genomic DNA. “cDNA”, as used herein, refers toa contiguous nucleotide sequence that encodes a polypeptide, and caninclude, but is not limited to a double-stranded DNA molecule generatedvia reverse transcription of an mRNA molecule.

“Isolated” or “purified” when used herein to describe a nucleic acidmolecule or nucleotide sequence, refers to a nucleic acid molecule ornucleotide sequence which is separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid molecule.Preferably, an “isolated” nucleic acid molecule is free of sequences(preferably protein encoding sequences) which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. Moreover, an “isolated” nucleic acid molecule, such asa cDNA molecule, can be substantially free of other cellular material,or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

“Isolated” or “purified” when used herein to describe a protein orbiologically active portion thereof (i.e., a polypeptide, peptide oramino acid fragment), refers to a protein or biologically active portionthereof substantially free of cellular material or other contaminatingproteins from the cell or tissue source from which the protein isderived, or substantially free of chemical precursors or other chemicalswhen chemically synthesized. A protein or biologically active portionthereof (i.e., a polypeptide, peptide or amino acid fragment) that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”).

The following abbreviations are also used herein throughout and have thefollowing meanings:

-   -   CTL's cytotoxic T lymphocytes    -   TH cells T helper cells    -   APC's antigen presenting cells    -   MHC major histocompatibility complex    -   IL-2 interleukin-2    -   IL-4 interleukin-4    -   IL-5 interleukin-5    -   IL-10 interleukin-10    -   IL-13 interleukin-13    -   IFN-γ interferon-gamma    -   TM transmembrane domain    -   ECD extracellular domain    -   CD cytoplasmic domain

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Nucleotide sequence of clone 103.1 of band 103 (SEQ ID NO:1).

FIG. 2. 103 gene products. This diagram illustrates the relationshipbetween the sequence encoded by band 103, 103 gene (also known as ST-2,T1 and Fit-1) products and the IL-1 receptor polypeptide structure. Theextracellular, transmembrane and cytoplasmic domains of the proteins arenoted, along with the amino acid residues marking the boundaries ofthese domains. (Adapted from Yanagisawa et al., 1993, FEBS Lett.318:83-87.)

FIG. 3A-B. A) A nucleotide sequence encoding a secreted form of themurine 103 gene product is depicted (SEQ ID NO:2; GenBank Accession No.E07714). B) An amino acid sequence of a secreted form of murine 103 isdepicted (SEQ ID NO:6; GenBank Accession No. P14719).

FIG. 4A-B. A) A nucleotide sequence encoding a transmembrane form of themurine 103 gene product is depicted (SEQ ID NO:3; GenBank Accession No.E08652). B) An amino acid sequence of a transmembrane form of murine 103is depicted (SEQ ID NO:7; GenBankAccession No. S29498). The signalsequence domain of this transmembrane product extends from about aminoacid residue 1 to 23 of SEQ ID NO:7; the extracellular domain of thistransmembrane form extends from about amino acid residue 24 to 342 ofSEQ ID NO:7; the transmembrane domain of this transmembrane form extendsfrom about amino acids 343 to 366 of SEQ ID NO:7; the cytoplasmic orintracellular domain of this transmembrane form extends from about aminoacid residues 367 to 567 of SEQ ID NO:7.

FIG. 5A-B. A) A nucleotide sequence encoding a transmembrane form of thehuman 103 gene is depicted (SEQ ID NO:4; GenBank Accession No.A13012701). 5B) An amino acid sequence of the transmembrane form of thehuman 103 gene is depicted (SEQ ID NO:8; GenBank Accession No.BAA82405). The signal sequence of this transmembrane form of the human103 gene extends from about amino acid residue 1 to 18 of SEQ ID NO:8;the extracellular domain of this transmembrane form extends from aboutamino acid residues 1 to 323 of SEQ ID NO:8; the transmembrane domain ofthis transmembrane form extends from about amino acid residues 324 to350 of SEQ ID NO:8; the cytoplasmic or intracellular domain of thistransmembrane form extends from about amino acid residues 351 to 556 ofSEQ ID NO:8; and the immunoglobulin (Ig)-like domains of thistransmembrane form extends from about amino acid residues 29-89, 126-183and 228-305.

FIG. 6A-B. A) A nucleotide sequence encoding a secreted form of thehuman 103 gene is depicted (SEQ ID NO:5; GenBankAccession No. D12763).B) An amino acid sequence of a secreted form of the human 103 gene isdepicted (SEQ ID NO:9; GenBankAccession No. BAA02233).

FIG. 7. A) The nucleotide sequence encoding a variant form of the human103 gene is depicted (SEQ ID NO:10; GenBank Accession No. AB029084). B)The amino acid sequence of a variant form of the human 103 gene isdepicted (SEQ ID NO:11; GenBank Accession No. BAA85894).

FIG. 8. The nucleotide sequence (SEQ ID NO:12) and predicted amino acidsequence (SEQ ID NO:13) of the novel human 103 gene referred to hereinas Athdc120c9. The signal sequence of the Athdc120c9 gene productextends from about amino acid residues 1 to 18 of the amino acidsequence. The Ig-like domain of the Athdc120c9 gene product extends fromabout amino acid residues 29 to 89 of the amino acid sequence.

FIG. 9. An alignment of the two forms of the murine 103 gene product(SEQ ID NO:6 and SEQ ID NO:7) using CLUSTAL W (1.74).

FIG. 10. An alignment of the four forms of the human 103 gene product(SEQ ID NO ID:8, SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:13) usingCLUSTAL W (1.74).

FIG. 11. An alignment of the human and murine forms of the 103 geneproduct (SEQ ID NO ID:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:6 and SEQ ID NO:7) using CLUSTAL W (1.74).

FIG. 12. Quantitative RT-PCR analysis of 103 gene expression inpolarized populations of murine TH cells. RNA samples were harvestedfrom cultured T cell populations 24 hours after tertiary stimulationwith antigen. cDNA samples were PCR amplified and the products of thosereactions were electrophoresed on a 1% agarose gel and visualized byethidium bromide staining. 103 gene expression is shown in the upperpanel. γ-actin data, bottom panel, was included as a control fordifferences in sample quality. The numbers above each lane represent thedilution factors of each cDNA. The same cDNA samples were used for boththe 103 gene and the γ-actin amplifications.

FIG. 13. Northern blot analysis of 103 gene expression in representativemurine TH cell lines (TH2: CDC25, D10.G4, DAX; TH1: AE7.A, Dorris,D1.1). Clones were either unstimulated (−) or stimulated (+) for 6 hourswith plate-bound anti-CD3 antibody. Ten micrograms of total RNA wereloaded per lane. The positions of 18s and 28s ribosomal RNA are shown asreference markers.

FIG. 14. Northern blot analysis of 103 gene expression in T cell clonesand murine tissues. Lane 1: DAX cells, no stimulation; lane 2, AE7cells, stimulation; lane 3, AE7 cells, no stimulation; lane 4, D10.G4cells, stimulation; lane 5, D10.G4 cells, no stimulation; lane 6, brain;lane 7, heart; lane 8, lung; lane 9, spleen; lane 10, liver. Clones werestimulated with plate-bound anti-CD3 antibody for 6 hours. 7.5 and 10micrograms total RNA was used for each cell line and each tissue,respectively. a, b, and c arrows refer to RNA encoding full length (a)and truncated (b,c) forms of the 103 gene. The positions of 18s and 28sribosomal RNA markers are shown.

FIG. 15. RNAse protection analysis of 103 gene mRNA, illustratingregulation of 103 gene expression in murine TH cell clones. Lanes 2-6:β-actin protection; lanes 9-13: 103 gene protection; lanes 1 and 8:markers; lanes 2 and 9: unstimulated TH1 clones; lanes 3 and 10:stimulated TH1 clones; lanes 4 and 11: unstimulated TH2 clones; lanes 5and 12: stimulated TH2 clones; lanes 6 and 13: fully RNAse A digestedunprotected probe; lanes 7 and 14: probe alone, in absence of addedRNAse.

Expected Fragment Sizes:

β-actin protected probe: 250 nucleotides;

β-actin full length probe: 330 nucleotides;

103 gene long form fragment: 257 nucleotides;

103 gene short form fragment: 173 nucleotides;

103 gene full length probe: 329 nucleotides.

FIG. 16. Expression of the soluble and transmembrane forms of the human103 gene in following hematopoeitic cells was quantitatively determined:resting and phytohemaglutinin (PHA) activated peripheral bloodmononuclear cells (PBMC); resting and PHA activated CD3⁺ cells; CD4⁺ andCD8⁺ T cells; resting Th0, Th1 and Th2 cells; Th0, Th1 and Th2 cellsstimulated for 1, 6, 24 or 48 hours with anti-CD3 antibody; resting andlipopolysaccharide (LPS) activated CD 19⁺ B cells; CD 14⁺ cells;granulocytes; eosinophils; PBMC stimulated with IL-10 and IL-4; and PBMCstimulated with interferon-γ (IFN-γ) and tumor necrosis factor-α(TNF-α).

FIG. 17. Flow cytometry data demonstrates that the 3E10 mAb recognizesand binds to representative clones of the TH2 cell subpopulation(D10.G4; DAX), but not clones of the TH1 subtype (AE7; Dorris). Thegraphs in this figure present the results of the flow cytometry analysesby depicting the number of cells exhibiting a given level offluorescence. Staining above background levels representsantigen-specific binding and, therefore, the presence of cell surface103 gene product. The further to the right the peaks are shifted, thegreater the staining intensity, and therefore antibody binding,exhibited by a cell population.

FIG. 18. Analysis of the cytokine profile in mouse BAL. The datapresented in this figure reveals high levels of IL-4, IL-5, IL-6, IL-10and IL-13 in TH2 recipient OVA challenged mice (closed bars). There wasno detectable TH2 cytokines in the BAL fluid of mice that received TH2cells and were not exposed to ovalbumin. Pretreatment with 3E10 mAbresulted in a dramatic reduction in IL-4, IL-5, IL-6 and IL-13, but hadno effect on IL-10 levels in the BAL (open bars). OVA challenge of TH1recipient mice resulted in high levels of IFN-γ in the BAL fluid (closedbars) that was not inhibited by 3E10 mAb (open bars). Data are shown asthe mean±sem of 5-6 animals.

FIGS. 19A-B. Anti-103 gene product mAb inhibits TH2 mediated allergiclung inflammation. A) Analysis of the number of eosinophils in the BAL;B) analysis of the number of lymphocytes in the BAL. The number ofOVA-specific TH2 cells in dispersed lung tissue as described (Cohn, L.et al., 1997, J. Exp. Med. 186:1737-1747). Lymphocytes were stained withbiotinylated clonotypic TCR mAb KJ126 (Cohn, L. et al., 1997 J. Exp.Med. 186:1737-1747) followed by strepavidin-FITC and CD4-PE (Pharmingen,San Diego).

FIG. 20. Inhibition of airway hyperresponsiveness by anti-103 geneproduct mAb. OVA exposure in TH2 recipient mice resulted in airwayhyperresponsiveness (closed squares) compared to mice exposed to PBS(closed circles). Pretreatment with 103 gene product mAb inhibited OVAinduced BHR by 80% (open diamonds). The results are shown as the meanPenh±sem of n=5-6 and is representative of 2 separate experiments.

FIGS. 21A-B. Administration of 3E10 mAb or the 103/Ig fusion results insignificant decrease in hallmark symptoms of asthma. A) Animals weretreated with the anti-103 3E10 antibody (listed in the figure as “3E10mAB”). As a negative control, a set of animals was treated with anon-specific rat Ig antibody preparation. B) Animals were treated with103/Ig fusion protein (listed in the figure as “Ig Fus. Prot.”) as anegative control, a control set of animals were treated with anon-specific human IgG antibody preparation.

FIG. 22. Crosslinking of 103 gene product augments IL-4 and IL-5cytokine secretion. TH2 effector cells were activated with plate-boundCD3 (3 μg/ml, 2C11) and CD28 (37.51, 4 μg/ml, Pharmingen San Diego) and3E10 (20 μg/ml) for 48 hrs. IL-4 and IL-5 levels were measured in thesupernatant by ELISA. 3E10 mAb stimulation alone failed to induce TH2cell activation but augmented both anti-CD3 and anti-CD3+CD28 inducedcytokine production. Soluble 3E10 failed to have any effect on CD3/CD28mediated cytokine production. These data suggest that activation of 103gene product provides a stimulatory signal to TH2 cells. There was noeffect of the mAb on TH2 cell proliferation as revealed by ³H-thymidineincorporation. 3E10 mAb did not modify IFN-γ secretion from TH1 effectorcells stimulated under the same conditions.

FIGS. 23A-D. Flow cytometry data demonstrate that the 3E10 mAbrecognizes and binds to CD4+ cells cultured in conditions which promotedTH2 development (FIG. 23C), but not in naive CD4+ cells (FIG. 23B) or inCD4+ cells cultured in conditions which promote TH1 development (FIG.23D); the 3E10 mAb also failed to bind to splenocytes, indicating thatthe 103 gene product is expressed only on the surface of TH2 or TH2-likecells.

FIG. 24 shows cytokine (IL-4, IL-5 and IFN-γ) levels measured fromantigen restimulated CD4+ T cells differentiated with OVA peptide alone,in TH1 polarizing conditions (i.e., with IL-12 and anti-IL-4 mAb) or inTH2 polarizing conditions (i.e., with IL-4 and anti-IL-12 mAb) for fivedays in the presence of human-Ig (closed bars) or in the presence of the103-Ig fusion protein (open bars).

FIG. 25 depicts cytokine (IL-4, IL-5, and IFN-γ) production levelsmeasured in separate TH1 and TH2 effector populations activated withpeptide and mitomycin C-treated splenocytes in the presence of eitherhuman-Ig (100 μg/mL; open squares) 103-Ig fusion protein (1-100 μg/mL)or a control fusion protein, designated H1-Ig (open squares).

FIGS. 26A-C. Inhibition of cellular and humoral responses in an activeimmunization model; responses were measured in untreated OVA allergenexposed mice (closed columns), rat IgG1 treated (100 μg) mice (opencolumns), and 3E10 mAb treated (100 μg) mice (shaded columns); FIG. 26Ashows eosinophil counts; FIG. 26B shows measured IL-5 levels; and FIG.26C shows measured IgE levels in each of the three experiments.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the treatment (e.g., amelioration ofsymptoms), prognosis and diagnosis of immune disorders, especially THcell subpopulation-related disorders are described herein. The immunedisorders include, but are not limited to, chronic inflammatory diseaseand disorders (e.g., Crohn's disease, reactive arthritis, and Lymedisease), insulin-dependent diabetes, organ specific autoimmunity(including, e.g., multiple sclerosis, Hashimoto's thyroiditis andGrave's disease), contact dermatitis, psoriasis, graft rejection, graftversus host disease, sarcoidosis, atopic conditions (e.g., asthma andallergy including, but not limited to, allergic rhinitis andgastrointestinal allergies such as food allergies), eosinophilia,conjunctivitis, glomerular nephritis, systemic lupus erythematosus,scleroderma certain pathogen susceptibilities such as helminthic (e.g.,leishmaniasis) and certain viral infections (including HIV and bacterialinfections such as tuberculosis and lepromatous leprosy).

Specifically, the methods and compositions of the present invention canutilize a gene, referred to herein as the 103 gene or gene sequence, aswell as gene products of the 103 gene and/or modulators thereof, e.g.,antibodies which specifically bind to such 103 gene products. Certain103 gene and gene products are alternately referred to in the art asST2, T1 and Fit-1. See, for example, Klemenz, R. et al., 1989, Proc.Natl. Acad. Sci. USA 86:5708-5712; S. Tominaga, 1989, FEBS Lett.258:301-301; A. K. Werenskiold et al., 1989, Mol. Cell. Biol.9:5207-5214; S. Tominaga et al., 1992, Biochem. Biophys. Acta.1171:215-218; A. K. Werenskiold, 1992, Eur. J. Biochem. 204:1041-1047;K. Yanagisawa et al., 1993, FEBS Lett. 318:83-87; G. Bergers et al.,1994, EMBO J 13:1176-1188; S. Kumar, 1997, Biochem Biophys Res Commun235:474-478; S. Tominaga, 1994, Japanese Patent No. JP 1994178687-A 3)each of which is incorporated herein, by reference, in its entirety.

The present invention encompasses methods and compositions comprisingmurine 103 gene products and the nucleotide sequences encoding thosegene products. The murine 103 gene encodes at least two forms, a 337amino acid soluble or secreted form, otherwise referred to as murineST2, and a 567 amino acid transmembrane form, otherwise referred to asmurine ST2L (see, e.g., Tominaga et al., 1989, FEBS Lett. 258 (2):301-304; Yanagisawa et al., 1993, FEBS Lett. 318 (1): 83-87; Kumar, S.,1997, Biochem. Biophys. Res. Comm. 225:447-478). FIG. 3B depicts theamino acid sequence of a 337 amino acid secreted form of the murine 103gene product (SEQ ID NO:6) encoded by the nucleic acid sequence in FIG.3A (SEQ ID NO:2). FIG. 4B depicts the amino acid sequence of a 567 aminoacid transmembrane form of the murine 103 gene product (SEQ ID NO:7)encoded by the nucleic acid sequence shown in FIG. 4A (SEQ ID NO:3).FIG. 9 depicts an alignment of the two forms of the murine gene product.The soluble form of the murine 103 gene product (SEQ ID NO:6) consistsof the first 328 amino acid residues of the transmembrane form of themurine 103 gene product (SEQ ID NO:7) and 9 different amino acidresidues.

The present invention also encompasses methods and compositionscomprising human 103 gene products and the nucleotide sequences encodingthose gene products. Three forms of human 103 gene products have beendescribed, a 323 amino acid soluble or secreted form, otherwise referredto as human ST2, a 556 transmembrane form, otherwise referred to ashuman ST2L, and a 259 amino acid form, otherwise referred to as ST2V(see, e.g., Tominaga et al., 1992, Biochem. Biophys. Res. Comm.1171:215-218; Tominaga et al., 1999, Biochem. Biophys. Res. Comm.264:14-18). FIG. 5B depicts the amino acid sequence of a 556 amino acidtransmembrane form of the human 103 gene product (SEQ ID NO:8) encodedby the nucleic acid sequence in FIG. 5A (SEQ ID NO:4). FIG. 6B depictsthe amino acid sequence of a 323 amino acid secreted form of the human103 gene product (SEQ ID NO:9) encoded by the nucleic acid sequenceshown in FIG. 6A (SEQ ID NO:5). FIG. 7B depicts the amino acid sequenceof a 259 amino acid variant form of the human 103 gene product (SEQ IDNO:11) encoded by the nucleic acid sequence shown in FIG. 7A (SEQ IDNO:10). The transmembrane form of the human 103 gene product (SEQ IDNO:8) consists of the first 323 amino acids of the secreted form of the103 gene product (SEQ ID NO:9). The 259 amino acid form in of the human103 gene product (SEQ ID NO:11) consists of the first 203 amino acids ofthe secreted form of the 103 gene product (SEQ ID NO:9). The 259 aminoacid form of the human 103 gene product (SEQ ID NO:11) has a hydrophobictail and lacks the third Ig-like domain found in the secreted form ofthe human 103 gene product (SEQ ID NO:9).

At least one allelic variant of the secreted form of the human 103 geneexists. The nucleotide sequence of this allelic variant differs from thenucleotide sequence depicted in FIG. 6A (SEQ ID NO:5) in that nucleicacid number 1130 of the allelic variant sequence is a guanine (G) ratherthan an adenine (A) (see, in particular, the nucleotide sequencedisclosed in GenBank Accession No. E07716). However, as will be apparentto one skilled in the art, because this single nucleotide variation (orpolymorphism) is located in the non-coding, 3′ untranslated region (UTR)of the gene sequence, this allelic variant encodes the same secretedform of a human 103 gene product as does the nucleotide sequence of FIG.6A (SEQ ID NO:5).

The invention also provides a novel human 103 gene product, disclosedherein for the first time. In particular, FIG. 8 depicts the amino acidsequence (SEQ ID NO:13) of the novel Athdc120c9 gene product, which isencoded by a nucleotide sequence comprising the sequence in the SEQ IDNO:12. In particular, this novel form of a human 103 gene productconsists of the first 150 amino acid residues (i.e., amino acid residues1-150) of the human 103 gene products depicted in FIGS. 5B and 6B (SEQID NOS:8 and 9, respectively) and 8 different amino acid residues. Thesignal sequence of the Athdc120c9 gene product extends from about aminoacid residues 1 to 18 of the amino acid sequence. In one embodiment, anisolated polypeptide comprises amino acid residues 19 to 158 in SEQ IDNO:8 (FIG. 8). FIG. 10 depicts an alignment of the four forms of human103 gene products. FIG. 11 depicts an alignment of the murine and humanforms of 103 gene products.

Domains of the 103 gene products (e.g., the domains depicted in FIG. 2)are also among the 103 gene products which can be used in the methodsand compositions of the present invention. Likewise, nucleotidesequences which encode any one or more of these domains can also be usedin the methods and compositions of this invention.

Exemplary domains of the 103 gene products include a signal sequencedomain (SS), an extracellular domain (ECD), a transmembrane domain (TM)and a cytoplasmic domain (CD) which is also referred to herein as theintracellular domain. FIG. 2 depicts a schematic alignment of secretedand transmembrane forms of the murine 103 gene product (labeled ST2 andST2L, respectively) as well as another, related protein: the murineinterleukin-1 receptor type 1 protein (IL1-R1). The extracellular,transmembrane and cytoplasmic domains of these proteins are indicated inFIG. 2. In one embodiment, the transmembrane form of the murine 103 geneproduct has a signal sequence domain corresponding to about amino acidresidue 1 to about amino acid residue 23 of the amino acid sequencedepicted in FIG. 4B (SEQ ID NO:7), an extracellular domain correspondingto about amino acid residue 24 to about amino acid residue 342 of theamino acid sequence depicted in FIG. 4B (SEQ ID NO:7), a transmembranedomain corresponding to about amino acid residue 343 to about amino acidresidue 366 of SEQ ID NO:7, and a cytoplasmic or intracellular domaincorresponding to about amino acid residue 367 to about amino acidresidue 567 of the amino acid sequence in FIG. 4B (SEQ ID NO:7). Inanother embodiment, the transmembrane form of the murine 103 geneproduct has a signal sequence corresponding to about amino acid residues1 to 23 of SEQ ID NO:7, an extracellular domain corresponding to aboutamino acids 24 to 333 of SEQ ID NO:7, a transmembrane domaincorresponding to about amino acid residues 334 to 355 of SEQ ID NO:7,and a cytoplasmic domain corresponding to amino acid residues 356 to 567of SEQ ID NO:7.

Similar domains are known to exists in the human 103 gene product. Inone embodiment, the transmembrane form of the human 103 gene product hasa signal sequence domain corresponding to about amino acid residue 1 toabout amino acid residue 18 of the amino acid sequence depicted in FIG.5B (SEQ ID NO:8), an extracellular domain corresponding to about aminoacid residue 19 to about amino acid residue 323 of the amino acidsequence depicted in FIG. 5B (SEQ ID NO:8), a transmembrane domaincorresponding to about amino acid residue 324 to about amino acidresidue 350, and a cytoplasmic or intracellular domain corresponding toabout amino acid residue 351 to 556 of the amino acid sequence in FIG.5B (SEQ ID NO:8). In another embodiment, the transmembrane form of thehuman 103 gene product has a signal sequence corresponding to aboutamino acid residue 1 to about amino acid residue 18 of the amino acidsequence of SEQ ID NO:8, an extracellular domain corresponding to aboutamino acid residue 19 to about amino acid residue 323 of the amino acidsequence depicted of SEQ ID NO:8, a transmembrane corresponding to aboutamino acid residue 324 to about amino acid residue 350 of the amino acidsequence of SEQ ID NO:8, and a cytoplasmic domain corresponding to aboutamino acid residue 351 to amino acid residue 556 of SEQ ID NO:8.

Other domains will also be apparent to those skilled in the art. Forexample, the 103 gene products of the invention also containimmunoglobulin (Ig)-like domains. An Ig domain typically has theconsensus the following consensus sequence, beginning at about 1 to 15amino acid residues, more preferably about 3 to 10 amino acid residues,and most preferably about 5 amino acid residues from the C-terminal endof the domain: [FY]-Xaa-C-Xaa-[VA]-Xaa-H-COO-, wherein [FY] is either aphenylalanine or a tyrosine residue (preferably tyrosine), where “Xaa”is any amino acid, C is a cysteine residue, [VA] is either valine or analanine residue (preferably alanine), and COO- is the C-terminus of thedomain. The secreted form of human 103 gene product depicted in FIG. 6B(SEQ ID NO:9) has at least three Ig-like domains corresponding to aboutamino acid residue 29 to about amino acid residue 89, about amino acidresidue 126 to about amino acid residue 183, and about amino acidresidue 228 to about amino acid residue 305 of the amino acid sequencein FIG. 6B (SEQ ID NO:9). The Ig-like domains corresponding to aboutamino acid 29 to about amino acid residue 89 and about amino acidresidue 228 to about amino acid residue 305 of the amino acid sequencedepicted in FIG. 6B (SEQ ID NO:9) have the following consensus sequence,beginning at about 5 amino acid residues from the C-terminal end of thedomain: [FY]-Xaa-C-Xaa-[VA]-COO-, wherein [FY] is either a phenylalanineor a tyrosine residue (preferably tyrosine), where “Xaa” is any aminoacid, C is a cysteine residue, [VA] is either valine or an alanineresidue (preferably alanine), and COO- is the C-terminus of the domain.The Ig-like domain corresponding to about amino acid residue 126 toabout amino acid residue 183 the amino acid sequence depicted in FIG. 6B(SEQ ID NO:9) has the following consensus sequence, beginning at about 5amino acid residues from the C-terminal end of the domain:[FY]-Xaa-C-Xaa-COO-, wherein [FY] is either a phenylalanine or atyrosine residue (preferably tyrosine), where “Xaa” is any amino acid, Cis a cysteine residue, and COO- is the C-terminus of the domain. Asnoted above, the novel variant of the 103 gene product disclosed herein(i.e., Athdc120c9; FIG. 8, SEQ ID NO:12) consists of the first 150 aminoacid residues of the secreted form of human 103 gene product shown inFIG. 8B (SEQ ID NO:13) and 8 different amino acid residues. Thus, as oneskilled in the art readily appreciates, the Athdc120c9 gene product alsohas an Ig-like domain corresponding to about amino acid residue 29 toabout amino acid residues 89 of the amino acid sequence depicted in FIG.8 (SEQ ID NO:13). The Athdc120c9 gene product also has a signal sequencedomain corresponding to amino acid residue 1 to about amino acid residue18 of the amino acid sequence depicted in FIG. 8 (SEQ ID NO:13).

Other exemplary domains of the 103 gene products of the inventioninclude, but are not limited to, ligand binding domains. A skilledartisan can readily identify a ligand binding domain of a 103 geneproduct, e.g., by preparing antibodies to particular epitopes of the 103gene product, according to the methods described and demonstrated, e.g.,in Section 5.3 and in the Example presented in Section 6.8, below. Oneskilled in the art will readily appreciate that the amino acid residuescorresponding to epitopes that produce antibodies inhibiting the bindingof the 103 gene product to a ligand will correspond to ligand bindingdomains of that 103 gene product.

Functionally equivalent forms of each of these domains will be apparentto those skilled in the art in other forms of the 103 gene productsdescribed herein, including human forms of the 103 gene product.

The invention is based, in part, on the discovery that the 103 gene isexpressed, in vivo, in a tightly controlled TH2 specific manner, andthat the 103 gene product is an important molecule in signalingTH2-mediated immune responses. Thus, compounds such as natural ligands,derivatives of natural ligands and antibodies that specifically bind tothe 103 gene product can be utilized to modulate (e.g., reduce) thenumber of TH2 and/or TH2-like cells present, for example, by physicallyseparating such cells away from other cells in a population or,alternatively, by targeting the specific destruction of TH2 and/orTH2-like cells, or by inhibiting the proliferation of such TH2 and/orTH2-like cells. Additionally, compounds such as 103 gene sequences, 103gene products or anti-103 antibodies (i.e., antibodies that specificallybind to 103 gene products) can be utilized to reduce the level of TH2cell activity and/or to cause a reduction in the level of TH2 cellcytokine production (e.g, reduce the level of production of cytokines,such as IL-4, that are associated with TH2 or TH2-like cellsubpopulations and/or with TH2 or TH2-like cell subpopulation activity).For example, IL-4 produced by the TH2 cell subpopulation stimulates Bcells which, in turn, produce IgE-type antibodies. Thus conditions thatinvolve an inappropriate IgE immune response, including but not limitedto the symptoms which accompany atopic conditions such as allergy and/orasthma, can be treated and/or ameliorated by reducing IL-4 levels, e.g.,by using the methods of the present invention to reduce TH2 cellactivity.

The 103 gene is also expressed in human mast cells, as demonstrated inthe Example presented in Section 6.5, below. Thus, the above-describedcompositions (e.g., natural ligands, derivatives of natural ligands,small molecules and antibodies that specifically bind to the 103 geneproduct) can also be utilized to modulate the number of mast cellspresent and/or to modulate the amount of mast cell activity or mast cellcytokine production (e.g., from the degranulation of mast cells). Thusconditions, including atopic conditions such as asthma and allergy, thatinvolve or are mediated by mast cell activity (often in addition to TH2or TH2-like activity) can be treated by using the methods andcompositions of the invention to target mast cells and/or mast cellactivity as well as (or instead of) TH2 cells and/or TH2 cell activity.

The 103 gene and nucleotide sequences of the invention are described, indetail, in Section 5.1, below. Further, the gene products of the 103gene are described herein in Section 5.2, and antibodies to such geneproducts are described in Section 5.3. Methods for using the 103 genes,their gene products and anti-103 antibodies and/or modulators of 103gene expression or 103 gene product activity are also described herein.

In particular, methods for the identification of compounds whichmodulate the expression of genes, such as the 103 gene, involved in (a)TH cell subpopulation-related disorders, and/or (b) the differentiationand effector function of TH cell subpopulations are presented in Section5.4 The methods include both in vitro assays (described in Section5.4:1) and in vivo assays (e.g., cell and animal based models of variousTH cell subpopulation-related disorders, including the models describedin Section 5.4.4). Compositions and methods for the treatment of immunedisorders are also described below, in Section 5.5. Pharmaceuticalcompositions for use, e.g., in the diagnostic and treatment methods ofthe invention, are described in Section 5.6, as well as methods foradministering such compositions. Methods for the prognostic anddiagnostic evaluation of various TH cell subpopulation-relateddisorders, for the identification of subjects exhibiting apredisposition to such disorders, and for monitoring the efficacy ofcompounds used in clinical trials are described in Section 5.7.

The invention is demonstrated by way of several specific examplespresented in Section 6. These examples are presented by way ofillustration of the methods described in this section, and are notlimiting of that description in any way. Indeed, many modifications andvariations of the present invention can be made without departing fromits spirit and scope, as will be apparent to those skilled in the art.The specific embodiments described herein are offered by way of exampleonly, and the invention is to be limited only by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled.

5.1. THE 103 GENE

The 103 gene, which is also known as T1, ST2 or Fit-1, is describedherein. As used herein, 103 gene or gene sequence refers to: (a) atleast one of the nucleotide sequences and/or fragments thereof that aredepicted herein FIGS. 1, 3A, 4A, 5A, 6A, 7A and 8 (SEQ ID NOS:1-5, 10and 12); (b) any nucleotide sequence or fragment thereof that encodesthe amino acid sequence encoded by one of the nucleotide sequences thatare depicted in FIGS. 1, 3A, 4A, 5A, 6A, 7A and 8 (SEQ ID NOS:1-5, 10and 12); (c) any nucleotide sequence that hybridizes to the complementof one of the coding nucleotide sequences depicted herein in FIGS. 1,3A, 4A, 5A, 6A, 7A and 8 (SEQ ID NOS:1-5, 10 and 12) under stringentconditions, e.g., hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C., or hybridization tofilter-bound DNA in 0.5 M sodium pyrophosphate/7% SDS at about 65° C.followed by one or more washes in 0.2×SSC/1% SDS at about 42-55° C., orunder other stringent hybridization conditions which are known to thoseof skill in the art (see, for example, Ausubel, F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3); (d) any nucleotide sequence that hybridizes tothe complement of one of the coding nucleotide sequences depicted hereinin FIGS. 1, 3A, 4A, 5A, 6A, 7A and 8 (SEQ ID NOS:1-5, 10 and 12) underhighly stringent conditions, e.g., hybridization to filter-bound nucleicacid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C., or hybridization to filter-bound DNAin 0.5 M sodium pyrophosphate/7% SDS at about 65° C. followed by one ormore washes in 0.2×SSC/1% SDS at about 68° C., or under other stringenthybridization conditions which are known to those of skill in the art(see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocolsin Molecular Biology, Vol. I, Green Publishing Associates, Inc. and JohnWiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3), includingsuch other hybridization conditions as those described herein; and (e)the complement of any of the 103 genes or gene sequences recited in(a)-(d) above.

Preferably, the nucleic acid molecules that hybridize to the complementsof the 103 gene sequence disclosed herein are the same length or aboutthe same length as the 103 gene sequence disclosed herein (i.e., about4989, 1011, 1833, 1357 or 1210 nucleic acids in length) and/or alsoencode gene products, e.g., gene products that are the same length orabout the same length as a 103 gene product encoded by a nucleotidesequence of (a) above (i.e., 567, 556, 337, 328 or 158 amino acidresidues in length) and/or are functionally equivalent to a 103 geneproduct encoded by a nucleotide sequence of (a), above. “Functionallyequivalent,” as the term is used herein, can refer to, in certainembodiments, a gene product (e.g., a polypeptide) capable of exhibitinga substantially similar in vivo activity as an endogenous 103 geneproduct encoded by one or more of the above-recited 103 gene sequences.Alternatively, and in certain other embodiments, as when utilized aspart of assays such as those described hereinbelow (e.g., in Section5.4), “functionally equivalent” can refer to peptides or other moleculescapable of interacting with other cellular or extracellular molecules ina manner substantially similar to the way in which the correspondingportion of the endogenous 103 gene product would. Functionallyequivalent gene products can therefore include naturally occurring 103gene products present in the same or different species. Functionallyequivalent 103 gene products also include gene products that retain atleast one of the biological activities of a 103 gene product describedabove (e.g., which is encoded by the coding sequences depicted herein inFIGS. 1, 3A, 4A, 5A, 6A, 7A and 8; SEQ ID NOS:1-5, 10 and 12). Thefunctionally equivalent 103 gene products of the invention also includegene products which are recognized by and bind to antibodies (polyclonalor monoclonal) directed against one or more of 103 gene productsdescribed above (e.g., which are encoded by the coding sequencesdepicted herein in FIGS. 1, 3A, 4A, 5A, 6A, 7A and 8; SEQ ID NOS:1-5, 10and 12).

In a preferred embodiment, an isolated nucleic acid molecule encodes apolypeptide comprising amino acid residues 150 to 158 of SEQ ID NO:13and the nucleic acid molecule hybridizes under stringent conditions(i.e., highly or less stringent conditions defined above) to thecomplement of a nucleic acid molecule that encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:13. In another preferredembodiment, an isolated nucleic acid molecule encodes a polypeptidecomprising amino acid residues 150 to 158 of SEQ ID NO:13 and thenucleic acid molecule hybridizes under stringent conditions (i.e.,highly or less stringent conditions defined above) to the complement ofthe nucleotide sequence of SEQ ID NO:12.

Further, and as those skilled in the art readily appreciate, an aminoacid sequence encoded by a given nucleic acid sequence may also beencoded by a number of “degenerate” nucleic acid sequence which areapparent to those skilled in the art. Thus, the 103 gene sequences ofthe present invention also include degenerate variants of the sequencesdescribed in (a) through (d), above.

The 103 gene nucleotide sequences of the invention also encompass: (a)nucleotides that encode a mammalian 103 gene product, including thehuman and murine 103 gene products depicted herein in FIGS. 3B, 4B, 5B,6B, 7B and 8 (SEQ ID NOS:6-9, 11 and 13); (b) nucleotides that encodeportions of a 103 gene product that corresponds to one or more of itsfunctional domains including, but not limited to, a signal sequencedomain, an extracellular domain (ECD), a transmembrane domain (TM), acytoplasmic domain (CD; also referred to herein as an intracellulardomain) an immunoglobulin (Ig) domain and one or more ligand-bindingdomains; (c) nucleotide sequences that encode one or more splicevariants of a 103 gene product including, for example, sequences thatencode a splice variant of a 103 gene product; and (d) nucleotidesequences that encode mutants of a 103 gene product in which all or partof one of its domains is deleted or altered including, but not limitedto, mutants which encode soluble forms of the 103 gene product in whichall or a portion of the TM domain is deleted, and nonfunctionalreceptors in which all or a portion of a CD is deleted.

The 103 gene nucleotide sequences of the invention still further includenucleotide sequences that encode fusion proteins, such as IgFc fusionproteins, containing any one or more of the 103 gene products describedin (a)-(d) supra fused to another polypeptide. A fusion proteincomprises all or part (preferably biologically active) of a polypeptideencoded by a 103 nucleotide sequence operably linked to a heterologouspolypeptide (i.e., a polypeptide other than the same polypeptide of theinvention). Preferably, a fusion protein comprises the polypeptide inSEQ ID NO:13 or a fragment thereof which includes the carboxy-terminusof the polypeptide and a heterologous polypeptide.

The 103 gene nucleotide sequences of the invention still further includenucleotide sequences corresponding to the above described 103 genenucleotide sequences (i.e., the sequences described in (a)-(d) above andfusion proteins thereof) wherein one or more of the exons or fragmentsthereof, have been deleted.

Still further, the 103 gene nucleotide sequences of the invention alsoinclude nucleotide sequence that have at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 98% or more nucleotide sequence identity to one or more of the103 gene nucleotide sequences of (a)-(d) above. The 103 gene nucleotidesequences of the invention also include nucleotide sequences encodingpolypeptides that have at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%or more amino acid sequence identity to one or more of the polypeptidesencoded by any of the 103 gene nucleotide sequences of (a)-(e) above.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical overlapping positions/total # of positions×100%). In oneembodiment, the two sequences are the same length.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul(1993) Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al.,1990, J. Mol. Biol. 215:403-0. BLAST nucleotide searches can beperformed with the NBLAST nucleotide program parameters set, e.g., forscore=100, wordlength=12 to obtain nucleotide sequences homologous to anucleic acid molecules of the present invention. BLAST protein searchescan be performed with the XBLAST program parameters set, e.g., toscore-50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule of the present invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively,PSI-BLAST can be used to perform an iterated search which detectsdistant relationships between molecules (Id.). When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., of XBLAST and NBLAST) can be used Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, (1988)CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM 120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

The methods and compositions of the invention also encompass nucleicacid molecules, preferably DNA molecules, that hybridize to and aretherefore the complements of the 103 gene nucleotide sequences (a)through (e) in the preceding paragraph. Such hybridization conditionscan be highly stringent or less highly stringent, as described above.The nucleic acid molecules of the invention that hybridize to the abovedescribed DNA sequences include oligodeoxyoligonucleotides (“oligos”)which hybridize under highly stringent or stringent conditions to theDNA sequences (a) through (d) in the preceding paragraph. In general,for oligos between 14 and 70 nucleotides in length the meltingtemperature (Tm) is calculated using the formula: Tm(° C.)=81.5+16.6(log[monovalent cations (molar)]+0.41 (% G+C)−(500/N), where N is the lengthof the probe. If the hybridization is carried out in a solutioncontaining formamide, the melting temperature may be calculated usingthe equation: Tm(° C.)=81.5+16.6(log [monovalent cations(molar)])+0.41(% G+C)−(0.61% formamide)−(500/N) where N is the length ofthe probe. In general, hybridization is carried out at about 20-25degrees below Tm (for DNA-DNA hybrids) or about 10-15 degrees below Tm(for RNA-DNA hybrids). Other exemplary highly stringent conditions mayrefer, e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos).

These nucleic acid molecules can be used in the methods or compositionsof the invention, e.g., as 103 gene antisense molecules which areuseful, for example, in 103 gene regulation. The sequences can also beused as antisense primers, e.g., in amplification reactions of 103 genenucleic acid sequence. Further, such complementary sequences can be usedas part of ribozyme and/or triple helix sequence, also useful for 103gene regulation. Still further, such molecules can be used as componentsof diagnostic methods whereby the presence of or predisposition to, animmune disorder (e.g., a TH cell subpopulation related disorder) can bedetected.

Fragments of the 103 gene and 103 gene nucleotide sequences of theinvention can be at least 10 nucleotides in length. In alternativeembodiments, the fragments can be about 20, 30, 40, 50, 60, 70, 80, 90,100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500or more contiguous nucleotides in length. Alternatively, the fragmentscan comprise sequences that encode at least 10, 20, 30, 40, 50, 100,150, 200, 250, 300, 350, 400, 450, 500 or more contiguous amino acidresidues of the 103 gene products. Fragments of the 103 gene nucleicacid molecules of the invention can also refer to exons or introns ofthe above described nucleic acid molecules, as well as portions of thecoding regions of such nucleic acid molecules that encode domains suchas extracellular domains (ECD), transmembrane domains (TM) andcytoplasmic domains (CD).

In specific embodiments, fragments of the 103 nucleotide sequencecomprise at least nucleotides 547 to 557 of SEQ ID NO:12, morepreferably at least nucleotides 530 to 557 of SEQ ID NO:12, at leastnucleotides 500 to 557 of SEQ ID NO:12, at least nucleotides 450 to 557of SEQ ID NO:12, at least nucleotides 400 to 557 of SEQ ID NO:12, atleast nucleotides 350 to 557 of SEQ ID NO:12, at least nucleotides 300to 557 of SEQ ED NO:12, at least nucleotides 250 to 557 of SEQ ID NO:12,at least nucleotides 200 to 557 of SEQ ID NO:12, at least nucleotides150 to 557 of SEQ ID NO:12, at least nucleotides 100 to 557 of SEQ IDNO:12, at least nucleotides 75 to 557 of SEQ ID NO:12, at leastnucleotides 50 to 557 of SEQ ID NO:12, or at least nucleotides 25 to 557of SEQ ID NO:12.

In other embodiments, fragments of the 103 nucleotide sequence compriseat least nucleotides 1200 to 1210 of SEQ ID NO:12, more preferably atleast nucleotides 1175 to 1210 of SEQ ID NO:12, at least nucleotides1150 to 1210 of SEQ ID NO:12, at least nucleotides 1125 to 1210 of SEQID NO:12, at least nucleotides 1100 to 1210 of SEQ ID NO:12, at leastnucleotides 1075 to 1210 of SEQ ID NO:12, at least nucleotides 1050 to1210 of SEQ ID NO:12, at least nucleotides 1000 to 1210 of SEQ ID NO:12,at least nucleotides 950 to 1210 of SEQ ID NO:12, at least nucleotides900 to 1210 of SEQ ID NO:12, at least nucleotides 850 to 1210 of SEQ IDNO:12, at least nucleotides 800 to 1210 of SEQ ID NO:12, at leastnucleotides 750 to 1210 of SEQ ID NO:12, at least nucleotides 700 to1210 of SEQ ID NO:12, at least nucleotides 650 to 1210 of SEQ ID NO:12,at least nucleotides 600 to 1210 of SEQ ID NO:12, at least nucleotides550 to 1210 of SEQ ID NO:12, at least nucleotides 500 to 1210 of SEQ IDNO:12, at least nucleotides 450 to 1210 of SEQ ID NO:12, at least 400 to1210 of SEQ ID NO:12, at least nucleotides 350 to 1210 of SEQ ID NO:12,at least nucleotides 300 to 1210 of SEQ ID NO:12, at least nucleotides250 to 1210 of SEQ ID NO:12, at least nucleotides 200 to 1210 of SEQ IDNO:12, at least nucleotides 150 to 1210 of SEQ ID NO:12, at leastnucleotides 100 to 1210 of SEQ ID NO:12, at least nucleotides 50 to 1210of SEQ ID NO:12, or at least nucleotides 25 to 1210 of SEQ ID NO:12.

In other embodiments, a polypeptide of the invention, e.g., a fragmentof a 103 polypeptide, comprises at least amino acid residues 148 to 158of SEQ ID NO:13, more preferably at least amino acid residues 125 to 158of SEQ ID NO:13, at least amino acid residues 100 to 158 of SEQ IDNO:13, at least amino acid residues 75 to 158 of SEQ ID NO:13, at leastamino acid residues 50 to 158 of SEQ ID NO:13, or at least amino acidresidues 25 to 158 of SEQ ID NO:13.

The methods and compositions of the invention also use, and thereforeencompass, (a) DNA vectors that contain any of the foregoing codingsequences and/or their complements (i.e., antisense); (b) DNA expressionvectors that contain any of the foregoing coding sequences operativelyassociated with a regulatory element that directs the expression of thecoding sequences; and (c) genetically engineered host cells that containany of the foregoing coding sequences operatively associated with aregulatory element, such as a heterologous regulatory element, thatdirects the expression of the coding sequences in the host cell. As usedherein, regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors. The invention includes fragments of any of the DNAsequences disclosed herein.

In addition to the gene sequences described above, homologs of thesegene sequences and/or full length coding sequences of these genes, ascan be present in the same or other species, can be identified andisolated, without undue experimentation, by molecular biologicaltechniques well known in the art. Further, there can exist genes atother genetic loci within the genome of the same species that encodeproteins which have extensive homology to one or more domains of suchgene products. These genes can also be identified via similartechniques.

For example, the isolated differentially expressed gene sequence can belabeled and used to screen a cDNA library constructed from mRNA obtainedfrom the organism of interest. Hybridization conditions should be of alower stringency when the cDNA library was derived from an organismdifferent from the type of organism from which the labeled sequence wasderived. cDNA screening can also identify clones derived fromalternatively spliced transcripts in the same or different species.Alternatively, the labeled fragment can be used to screen a genomiclibrary derived from the organism of interest, again, usingappropriately stringent conditions. Low stringency conditions will bewell known to those of skill in the art, and will vary predictablydepending on the specific organisms from which the library and thelabeled sequences are derived. For guidance regarding such conditionssee, for example, Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, (Green Publishing Associates andWiley Interscience, N.Y.).

Further, a previously unknown 103 gene sequence can be isolated byperforming PCR using two degenerate oligonucleotide primer poolsdesigned on the basis of nucleotide sequences within one or more of theabove described known 103 gene sequences. The template for the reactioncan be cDNA obtained by reverse transcription of mRNA prepared fromhuman or non-human cell lines or tissue known or suspected to express adifferentially expressed or pathway gene allele. The PCR product can besubcloned and sequenced to insure that the amplified sequences representthe sequences of a 103 gene nucleic acid sequence.

The PCR fragment can then be used to isolate a full length cDNA clone bya variety of methods. For example, the amplified fragment can be used toscreen a bacteriophage cDNA library. Alternatively, the labeled fragmentcan be used to screen a genomic library.

PCR technology can also be utilized to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source. A reversetranscription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid can then be “tailed” with guanines using a standardterminal transferase reaction, the hybrid can be digested with RNAase H,and second strand synthesis can then be primed with a poly-C primer.Thus, cDNA sequences upstream of the amplified fragment can easily beisolated. For a review of cloning strategies which can be used, seee.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual,Cold Springs Harbor Press, N.Y.; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, (Green Publishing Associates and WileyInterscience, N.Y.).

As will be appreciated by those skilled in the art, DNA sequencepolymorphisms of a 103 gene identified by the methods of the presentinvention will typically exist within a population of individualorganisms (e.g., within a human population). Such polymorphisms mayexist, for example, among individuals within a population due to naturalallelic variation. Such polymorphisms include ones that lead to changesin amino acid sequence. An allele is one of a group of genes whichoccurs alternatively at a given genetic locus. Accordingly, as usedherein, an “allelic variant” refers to a nucleotide sequence whichoccurs at a given locus or to a gene product encoded by the nucleotidesequence. Natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of a given gene.

Alternative alleles or allelic variants can be identified by sequencingthe gene of interest in a number of different individuals. This can bereadily carried out by using hybridization probes to identify the samegenetic locus in a variety of individuals. As used herein, the terms“gene” and “recombinant gene” refer to nucleic acid molecules comprisingan open reading frame encoding a polypeptide of the invention. The termcan further include nucleic acid molecules comprising upstream and/orexon/intron sequences and structure.

With respect to allelic variants of the 103 genes and gene products ofthe present invention, any and all nucleotide variations and/or aminoacid polymorphisms or variations that are the result of natural allelicvariation of the differentially expressed pathway genes and/or geneproducts are intended to be within the scope of the present invention.Such allelic variants include, but are not limited to, ones that do notalter the functional activity of a differentially expressed or pathwaygene product of the invention. Variants also include, but are notlimited to “mutant alleles.” As used herein, a “mutant allele” of adifferentially expressed or pathway gene or gene product of theinvention is an allelic variant which does alter the functional activityof the differentially expressed or pathway gene product encoded by thatgene.

In cases where the differentially expressed or pathway gene identifiedis the normal, or wild type, gene, this gene can be used to isolatemutant alleles of the gene. Such an isolation is preferable in processesand disorders which are known or suspected to have a genetic basis.Mutant alleles can be isolated, e.g., from individuals either known orsuspected to have a genotype which contributes to TH cellsubpopulation-disorder related symptoms. Mutant alleles and mutantallele products can then be utilized in the therapeutic and diagnosticassay systems described below.

A cDNA of a mutant gene can be isolated, for example, by using PCR, atechnique which is well known to those of skill in the art. In thiscase, the first cDNA strand can be synthesized by hybridizing a oligo-dToligonucleotide to mRNA isolated from tissue known to, or suspected ofbeing expressed in an individual putatively carrying the mutant allele,and by extending the new strand with reverse transcriptase. The secondstrand of the cDNA is then synthesized using an oligonucleotide thathybridizes specifically to the 5′ end of the normal gene. Using thesetwo primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant gene to that of the normal gene, the mutation(s)responsible for the loss or alteration of function of the mutant geneproduct can be ascertained.

Alternatively, a genomic or cDNA library can be constructed and screenedusing DNA or RNA, respectively, from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. The normal gene or any suitable fragmentthereof can then be labeled and used as a probed to identify thecorresponding mutant allele in the library. The clone containing thisgene can then be purified through methods routinely practiced in theart, and subjected to sequence analysis as described, above, in thisSection.

Additionally, an expression library can be constructed utilizing DNAisolated from or cDNA synthesized from a tissue known to or suspected ofexpressing the gene of interest in an individual suspected of or knownto carry the mutant allele. In this manner, gene products made by theputatively mutant tissue can be expressed and screened using standardantibody screening techniques in conjunction with antibodies raisedagainst the normal gene product, as described, below, in Section 5.3.(For screening techniques, see, for example, Harlow, E. and Lane, eds.,1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Press, ColdSpring Harbor.) In cases where the mutation results in an expressed geneproduct with altered function (e.g., as a result of a missensemutation), a polyclonal set of antibodies are likely to cross-react withthe mutant gene product. Library clones detected via their reaction withsuch labeled antibodies can be purified and subjected to sequenceanalysis as described supra in this Section.

Other allelic variants and/or mutant variants of the 103 genes of theinvention include single nucleotide polymorphisms (SNPs), includingbiallelic SNPs or biallelic markers which have two alleles, both ofwhich are present at a fairly high frequency in a population oforganisms. Conventional techniques for detecting SNPs include, e.g.,conventional dot blot analysis, single stranded conformationalpolymorphism (SSCP) analysis (see, e.g., Orita et al., 1989, Proc. Natl.Acad. Sci. USA 86:2766-2770), denaturing gradient gel electrophoresis(DGGE), heteroduplex analysis, mismatch cleavage detection, and otherroutine techniques well known in the art (see, e.g., Sheffield et al.,1989, Proc. Natl. Acad. Sci. 86:5855-5892; Grompe, 1993, Nature Genetics5:111-117). Alternative, preferred methods of detecting and mapping SNPsinvolve microsequencing techniques wherein an SNP site in a target DNAis detected by a single nucleotide primer extension reaction (see, e.g.,Goelet et al., PCT Publication No. WO 92/15712; Mundy, U.S. Pat. No.4,656,127; Vary and Diamond, U.S. Pat. No. 4,851,331; Cohen et al., PCTPublication No. WO 91/02087; Chee et al., PCT Publication No. Wo95/11995; Landegren et al., 1988, Science 241:1077-1080; Nicerson etal., 1990, Proc. Natl. Acad. Sci. 87:9823-8927; Pastinen et al., 1997,Genome Res. 7:606-614; Pastinen et al., 1996, Clin. Chem. 42:1391-1397;Jalanko et al., 1992, Clin. Chem 38:39-43; Shumaker et al., 1996, Hum.Mutation 7:346-354; Caskey et al., PCT Publication No. 95/00669).

5.2. 103 GENE PRODUCTS

The 103 gene products used and encompassed in the methods andcompositions of the present invention include those gene products (e.g.,proteins) that are encoded by the 103 gene sequences described inSection 5.1, above, such as, for example, the polypeptides depictedherein in FIGS. 3B, 4B, 5B, 6B, 7B and 8 (SEQ ID NOS:6-9, 11, and 13).In addition, however, the methods and compositions of the invention alsouse and encompass proteins and polypeptides that represent functionallyequivalent gene products. Such functionally equivalent gene productsinclude, but are not limited to, natural variants of the polypeptidesdepicted herein in FIGS. 3B, 4B, 5B, 6B, 7B and 8 (SEQ ID NOS:6-9, 11,and 13). Such equivalent 103 gene products can contain, e.g., deletions,additions or substitutions of amino acid residues within the amino acidsequences encoded by the 103 gene sequences described above in Section5.1, but which result in a silent change, thus producing a functionallyequivalent 103 gene product. Amino acid substitutions can be made on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity and/or the amphipathic nature of the residues involved.For example, nonpolar (i.e., hydrophobic) amino acid residues caninclude alanine (Ala or A), leucine (Leu or L), isoleucine (Ile or I),valine (Val or V), proline (Pro or P), phenylalanine (Phe or F),tryptophan (Trp or W) and methionine (Met or M); polar neutral aminoacid residues can include glycine (Gly or G), serine (Ser or S),threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y),asparagine (Asn or N) and glutamine (Gln or Q); positively charged(i.e., basic) amino acid residues can include arginine (Arg or R),lysine (Lys or K) and histidine (His or H); and negatively charged(i.e., acidic) amino acid residues can include aspartic acid (Asp or D)and glutamic acid (Glu or E).

“Functionally equivalent,” as the term is utilized herein, refers to apolypeptide capable of exhibiting a substantially similar in vivoactivity as the endogenous 103 gene product encoded by one or more ofthe 103 gene sequences described in Section 5.1, above. Alternatively,when utilized as part of assays described hereinbelow (e.g., in Section5.4), the term “functionally equivalent” can refer to peptides orpolypeptides that are capable of interacting with other cellular orextracellular molecules in a manner substantially similar to the way inwhich the corresponding portion of the endogenous 103 gene product wouldinteract with such other molecules. Preferably, the functionallyequivalent 103 gene products of the invention are also the same size orabout the same size as an endogenous 103 gene product encoded by one ormore of the 103 gene sequences described in Section 5.1, above (i.e.,567, 556, 337, 321, 259 or 158 amino acid residues in length).

Peptides and polypeptides corresponding to one or more domains of the103 gene products (e.g., TM, ECD, CD, Ig or ligand-binding domains),truncated or deleted 103 gene products (e.g., polypeptides in which oneor more domains of a 103 gene product are deleted) and fusion 103 geneproteins (e.g., proteins in which a full length or truncated or deleted103 gene product, or a peptide or polypeptide corresponding to one ormore domains of a 103 gene product is fused to an unrelated protein) arealso within the scope of the present invention. Such peptides andpolypeptides can be readily designed by those skilled in the art on thebasis of the differentially expressed or pathway gene nucleotide andamino acid sequences disclosed above in this Section and in Section 5.1.Exemplary fusion proteins can include, but are not limited to, IgFcfusion proteins which stabilize the 103 gene product and prolong itshalf-life in vivo. Other exemplary fusion proteins include fusions toany amino acid sequence that allows, e.g., the fusion protein to beanchored to a cell membrane, thereby allowing 103 gene polypeptides tobe exhibited on a cell surface; or fusions to an enzyme, to afluorescent protein or to a luminescent protein which can provide amarker function.

Other modifications of the 103 gene product coding sequences describedabove can be made to generate polypeptides that are better suited, e.g.,for expression, for scale up, etc. and a chosen host cell. For example,cysteine residues can be deleted or substituted with another amino acidin order to eliminate disulfide bridges; N-linked glycosylation sitescan be altered or eliminated to achieve, for example, expression of ahomogenous product that is more easily recovered and purified from yeasthosts known to hyperglycosylate N-linked sites. To such an end, avariety of amino acid substitutions at one or both of the first or thirdamino acid residue positions of any one or more of the glycosylationrecognition sequences (e.g., N-X-S or N-X-T) and/or an amino aciddeletion at the second position of any one or more such recognitionsequences will prevent glycosylation of the protein at the modifiedtripeptide sequence (see, e.g., Miyajima et al., 1986, EMBO J.5:1193-1197).

The differentially expressed or pathway gene products of the inventionpreferably comprise at least as many contiguous amino acid residues asare necessary to represent an epitope fragment (that is, for the geneproducts to be recognized by an antibody directed to the 103 geneproduct). For example, such protein fragments or peptides can compriseat least about 8 contiguous amino acid residues from a full lengthdifferentially expressed or pathway gene product. In alternativeembodiments, the protein fragments and peptides of the invention cancomprise about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250,300, 350, 400, 450 or more contiguous amino acid residues of a 103 geneproduct.

The 103 gene products used and encompassed in the methods andcompositions of the present invention also encompass amino acidsequences encoded by one or more of the above-described 103 genesequences of the invention wherein domains encoded by one or more exonsof those sequences, or fragments thereof, have been deleted. The 103gene products of the invention can still further comprise posttranslational modifications, including, but not limited to,glycosylations, acetylations and myrisalations.

The 103 gene products of the invention can be readily produced, e.g., bysynthetic techniques or be methods of recombinant DNA technology usingtechniques that are well known in the art. Thus, methods for preparingthe 103 gene products of the invention are discussed herein. First, thepolypeptides and peptides of the invention can be synthesized orprepared by techniques well known in the art. See, for example,Creighton, 1983, Proteins: Structures and Molecular Principles, W.H.Freeman and Co., N.Y., which is incorporated herein by reference in itsentirety. Peptides can, for example, be synthesized on a solid supportor in solution.

Alternatively, recombinant DNA methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining 103 gene protein coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.which is incorporated by reference herein in their entirety, andAusubel, 1989, supra. Alternatively, RNA capable of encoding 103 geneprotein sequences can be chemically synthesized using, for example,synthesizers. See, for example, the techniques described inOligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems can be utilized to expressthe 103 gene coding sequences of the invention. Such host-expressionsystems represent vehicles by which the coding sequences of interest canbe produced and subsequently purified, but also represent cells whichcan, when transformed or transfected with the appropriate nucleotidecoding sequences, exhibit the 103 gene protein of the invention in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophageDNA, plasmid DNA or cosmid DNA expression vectors containing 103 geneprotein coding sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing the 103gene protein coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing the103 gene protein coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing 103 gene protein codingsequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the 103 geneprotein being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of antibodies or to screenpeptide libraries, for example, vectors which direct the expression ofhigh levels of fusion protein products that are readily purified can bedesirable. Such vectors include, but are not limited, to the E. coliexpression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in whichthe 103 gene protein coding sequence can be ligated individually intothe vector in frame with the lacZ coding region so that a fusion proteinis produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509);and the like. pGEX vectors can also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene protein can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The 103 gene coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of 103 genecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed, (e.g., see Smith et al., 1983, J. Viol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the 103 gene coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing 103 gene protein in infected hosts, (e.g., See Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specific initiationsignals can also be required for efficient translation of inserted 103gene coding sequences. These signals include the ATG initiation codonand adjacent sequences. In cases where an entire 103 gene, including itsown initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals can be needed. However, in cases where only a portion of the 103gene coding sequence is inserted, exogenous translational controlsignals, including, perhaps, the ATG initiation codon, must be provided.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBittner et al., 1987, Methods in Enzymol. 153:516-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellswhich possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

As used herein, the term “host cell” refers not only to the particularsubject cell transfected with a nucleic acid molecule of the inventionbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the term“host cell” as used herein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe 103 gene protein can be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which express the 103 geneprotein. Such engineered cell lines can be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the 103 gene protein.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Proc. Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl.Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapinet al., 1981, J Mol. Biol. 150:1); and hygro, which confers resistanceto hygromycin (Santerre et al., 1984, Gene 30:147) genes.

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human cellslines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

When used as a component in assay systems such as those describedherein, the 103 gene protein can be labeled, either directly orindirectly, to facilitate detection of a complex formed between the 103gene protein and a test substance. Any of a variety of suitable labelingsystems can be used including but not limited to radioisotopes such as¹²⁵I; enzyme labelling systems that generate a detectable colorimetricsignal or light when exposed to substrate; and fluorescent labels.

Indirect labeling involves the use of a protein, such as a labeledantibody, which specifically binds to either a 103 gene product. Suchantibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by an Fabexpression library.

Where recombinant DNA technology is used to produce the 103 gene proteinfor such assay systems, it can be advantageous to engineer fusionproteins that can facilitate labeling (either direct or indirect),immobilization, solubility and/or detection.

Fusion proteins, which can facilitate solubility and/or expression, andcan increase the blood half-life of the protein, can include, but arenot limited to soluble Ig-tailed fusion proteins. Methods forengineering such soluble Ig-tailed fusion proteins are well known tothose of skill in the art. See, for example U.S. Pat. No. 5,116,964,which is incorporated herein by reference in its entirety. Further, inaddition to the Ig-region encoded by the IgG1 vector, the Fc portion ofthe Ig region utilized can be modified, by amino acid substitutions, toreduce complement activation and Fc binding. (See, e.g., European PatentNo. 239400 B1, Aug. 3, 1994). The 103 gene product contained within suchIg-tailed fusion proteins can comprise, for example, the 103 geneextracellular or secreted domain of the 103 gene product or portions(preferably ligand-binding portions) thereof. The example presented inSection 6.2 below describes the construction of an exemplary 103 geneproduct-Ig fusion protein.

5.3. ANTIBODIES SPECIFIC FOR 103 GENE PRODUCTS

Described herein are methods for the production of antibodies capable ofspecifically recognizing epitopes of one or more of the 103 geneproducts described in Section 5.2 above. Such antibodies can include,but are not limited to, polyclonal antibodies, monoclonal antibodies(mAbs), human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by aFab expression library, anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above. The Fc tails of suchantibodies can be modified to reduce complement activation and FcRbinding. (See, for example, European Patent No. 239400 B1, Aug. 3,1994).

For the production of antibodies to a 103 gene or gene product, varioushost animals can be immunized by injection with a 103 gene protein, or aportion thereof. Such host animals can include but are not limited torabbits, mice, and rats, to name but a few. Various adjuvants can beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, can be immunized by injection withdifferentially expressed or pathway gene product supplemented withadjuvants as also described above. The antibody titer in the immunizedanimal can be monitored over time by standard techniques, such as withan enzyme linked immunosorbent assay (ELISA) using immobilizedpolypeptide. If desired, the antibody molecules can be isolated from theanimal (e.g., from the blood) and further purified by well-knowntechniques, such as protein A chromatography to obtain the IgG fraction.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies can be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention can be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal antibody directed against a polypeptide of the invention canbe identified and isolated by screening a recombinant combinatorialimmunoglobulin library (e.g., an antibody phage display library) withthe polypeptide of interest. Kits for generating and screening phagedisplay libraries are commercially available (e.g., the PharmaciaRecombinant Phage Antibody System, Catalog No. 27-9400-01; and theStratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, U.S. Pat. No. 5,223,409; PCT Publication No. WO92/18619; PCT Publication No. WO 91/17271; PCT Publication No. WO92/20791; PCT Publication No. WO 92/15679; PCT Publication No. WO93/01288; PCT Publication No. WO 92/01047; PCT Publication No. WO92/09690; PCT Publication No. WO 90/02809; Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J 12:725-734.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are withinthe scope of the invention. A chimeric antibody is a molecule in whichdifferent portions are derived from different animal species, such asthose having a variable region derived from a murine mAb and a humanimmunoglobulin constant region. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarily determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in PCT Publication No. WO87/02671; European Patent Application 184,187; European PatentApplication 171,496; European Patent Application 173,494; PCTPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentApplication 125,023; Better et al. (1988) Science 240:1041-1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J.Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison (1985) Science 229:1202-1207; Oi et al. (1986)Bio/Techniques 4:214; U.S. Pat. No. 5,225,539; Jones et al. (1986)Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice which are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Monoclonal antibodies directed against theantigen can be obtained using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA and IgE antibodies.For an overview of this technology for producing human antibodies, seeLonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.), can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Bio/technology12:899-903).

Antibody fragments which recognize specific epitopes can be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,1989, Science _(—)246:1275-1281) to allow rapid and easy identificationof monoclonal Fab fragments with the desired specificity.

Antibodies of the present invention can also be generated using variousphage display methods known in the art. In phage display methods,functional antibody domains are displayed on the surface of phageparticles which carry the polynucleotide sequences encoding them. Phageexpressing an antigen binding domain that binds the antigen of interest(i.e., a 103 gene product) can be selected or identified with antigen,e.g., using labeled antigen or antigen bound or captured to a solidsurface or bead. Examples of phage display methods that can be used tomake the antibodies of the present invention include those disclosed inBrinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J.Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J.Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burtonet al., Advances in Immunology 57:191-280 (1994); PCT application No.PCT/GB91/01 134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/1 1236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Single chain antibodies of the invention can also be generated by knowntechniques including those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988).

Antibodies to the differentially expressed or pathway gene products can,in turn, be utilized to generate anti-idiotype antibodies that “mimic”such gene products, using techniques well known to those skilled in theart. (See, e.g., Greenspan & Bona, 1993, FASEB J 7(5):437-444; andNissinoff, 1991, J. Immunol. 147(8):2429-2438). For example, antibodieswhich bind to the ECD and competitively inhibit the binding of ligand tothe receptor can be used to generate anti-idiotypes that “mimic” the ECDand, therefore, bind and neutralize the ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens of TH cell subpopulation-related disorders.

Antibodies of the present invention may also be described or specifiedin terms of their binding affinity to a 103 gene product. Preferredbinding affinities include those with a dissociation constant or Kd lessthan 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M, or 10⁻¹⁵M.

The exemplary production of antibodies directed against the 103 geneproducts of the invention is described in the Examples presented inSection 6, below. Specifically, the Examples presented in Sections 6.4and 6.5, below, describe the production and characterization of mouseantibodies, including monoclonal antibodies, directed against theextracellular domains of murine and human 103 gene products,respectively. The Example presented in Section 6.7 describes theproduction of humanized monoclonal antibodies directed against theextracellular domain of a human 103 gene product. In one embodiment, amonoclonal antibody of the invention is produced by the hybridoma cloneM15 3F7.3, M15 2O3.1, M15 10F7.1, M15 1B4,1, M15 9F11.1 or M15 5A16.1.The invention also encompasses an antigen binding fragment of amonoclonal antibody produced by the hybridoma clone M15 3F7.3, M152O3.1, M15 10F7.1, M15 1B4.1, M15 9F11.1 or M15 5A16.1.

It is understood, therefore, that such antibodies are among theantibodies of the present invention. Likewise, one skilled in the artcan readily appreciate and will be able to prepare antibodies thatcompete with monoclonal antibodies, such as the specific monoclonalantibodies described in the Examples in Sections 6.4, 6.5 and 6.7, forbinding to a 103 gene product and which therefore bind to the sameepitope of the 103 gene product. Thus, such antibodies which recognizeand specifically bind to the same epitope of a 103 gene product, e.g.,as the monoclonal antibodies described herein, are also among theantibodies of the present invention. The present invention encompassesan isolated antibody that competes with the monoclonal antibody producedby hybridoma clone M15 3F7.3, M15 2O3.1, M15 10F7.1, M15 1B4.1, M159F11.1 or M15 5A16.1 for epitope binding. Antibodies that compete withmonoclonal antibodies of the invention can be identified in immunoassayssuch as a competition ELISA.

In one embodiment, the ability of an antibody to compete with amonoclonal antibody of the invention is determined in an assaycomprising: (a) incubating the antibody and the monoclonal antibody witha 103 polypeptide; and (b) measuring the binding of the monoclonalantibody to the 103 polypeptide, so that if less monoclonal antibodybinding is measured relative to that measured in the absence of theantibody, the antibody competes with the monoclonal antibody forbinding.

In accordance with this embodiment, the monoclonal antibody can belabeled with a detectable substance (e.g., an enzyme, a prostheticgroup, a fluorescent material, a luminescent materials, a bioluminescentmaterials, or a radioisotope) to facilitate measuring the binding of themonoclonal antibody to the 103 polypeptide in an ELISA. Alternatively, alabeled secondary antibody that only recognizes the monoclonal antibodycan be incubated with the 103 polypeptide following the incubation withthe monoclonal antibody to facilitate measuring the binding of themonoclonal antibody to the 103 polypeptide.

5.3.1. USE OF ANTIBODIES SPECIFIC FOR 103 GENE PRODUCTS

Antibodies directed against a 103 gene product or fragment thereof canbe used to detect the a 103 gene product (e.g., in a biological sample)in order to evaluate the abundance and pattern of expression of thepolypeptide. Antibodies directed to cell surface epitopes of a 103 geneproduct can be used to isolate a cell subpopulation of interest (e.g., aTH2 or TH2-like cell subpopulation, for either depletion or augmentationpurposes. Antibodies directed against a 103 gene product or fragmentthereof can also be used diagnostically to monitor protein levels of a103 gene product in tissue as part of a clinical testing procedure,e.g., to, for example, determine the efficacy of a given treatmentregimen (see, Section 5.7 below). Detection can be facilitated bycoupling the antibody to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, andradioactive materials. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S, ³²P, ³H or ⁹⁹Tc.

Further, antibodies directed against a 103 gene product or fragmentthereof can be used therapeutically to treat, prevent or inhibit animmune disorder described herein (e.g., asthma). Antibodies can also beused to alleviate one or more symptoms associated with an immunedisorder described herein. Antibodies can also be used to modify abiological activity of a 103 gene product. For example, antibodies canbe used to modulate TH cell subpopulation differentiation, maintenanceand/or effector function. To facilitate or enhance its therapeuticeffect, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion. A cytotoxin orcytotoxic agent includes any agent that is detrimental to cells.Examples include paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol, andpuromycin and analogs or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

An antibody can also be conjugated to a drug moiety, which is notlimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, α-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), tumor necrosis factor (“TNF”)-α, TNF-β,interferon (“IFN”)-γ, granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating a therapeutic moiety to antibodies are wellknown, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can beused as a therapeutic that is administered alone or in combination withchemotherapeutic agents.

5.4. SCREENING ASSAYS FOR COMPOUNDS THAT INTERACT WITH THE 103 GENEPRODUCT

The following assays are designed to identify compounds that bind totarget gene products, bind to other cellular proteins that interact withthe 103 gene product, and to compounds that interfere with theinteraction of the target gene product with other cellular proteins. Forexample such techniques can identify ligands for a 103 gene product. Acompound which binds a 103 gene product (a 103 gene product ligand, forexample) can, e.g., be tested for an ability to ameliorate symptoms ofTH2 or TH2-like related disorders such as asthma or allergy. Any suchbinding compound can also act as a marker for the presence of TH cellsubpopulations. Thus, for example, a compound which binds the 103 geneproduct can act as a marker, for example a diagnostic marker, for TH2 orTH2-like cells, e.g., for TH2 or TH2-like cell differentiation.

Binding compounds can include, but are not limited to, other cellularproteins. Binding compounds can also include, but are not limited to,peptides such as, for example, soluble peptides, including, but notlimited to, Ig-tailed fusion peptides, comprising, for example,extracellular portions of 103 gene product transmembrane receptors, andmembers of random peptide libraries (see, e.g., Lam et al., 1991, Nature354:82-84; Houghten et al., 1991, Nature 354:84-86) made of D- and/orL-configuration amino acids, phosphopeptides (including but not limitedto members of random or partially degenerate, directed phosphopeptidelibraries; see, e.g., Songyang et al., 1993, Cell 72:767-778),antibodies (including, but not limited to polyclonal, monoclonal, human,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules. In thecase of receptor-type target molecules, such compounds can includeorganic molecules (e.g., peptidomimetics) that bind to the ECD andeither mimic the activity triggered by the natural ligand (i.e.,agonists); as well as peptides, antibodies or fragments thereof, andother organic compounds that mimic the ECD (or a portion thereof) andbind to a “neutralize” natural ligand.

Computer modelling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate 103 gene expression or activity. Having identified such acompound or composition, the active sites or regions are preferablyidentified. In the case of compounds affecting receptor molecules, suchactive sites might typically be ligand binding sites, such as theinteraction domains of ligand with receptor itself. The active site canbe identified using methods known in the art including, for example,from the amino acid sequences of peptides, from the nucleotide sequencesof nucleic acids, or from study of complexes of the relevant compound orcomposition with its natural ligand. In the latter case, chemical orX-ray crystallographic methods can be used to find the active site byfinding where on the factor the complexed ligand is found.

The three dimensional geometric structure of the active site is thenpreferably determined. This can be done by known methods, includingX-ray crystallography, which can determine a complete molecularstructure. Solid or liquid phase NMR can also be used to determinecertain intra-molecular distances within the active site and/or in theligand binding complex. Any other experimental method of structuredetermination can be used to obtain partial or complete geometricstructures. The geometric structures may be measured with a complexedligand, natural or artificial, which may increase the accuracy of theactive site structure determined.

Methods of computer based numerical modelling can be used to completethe structure (e.g., in embodiments wherein an incomplete orinsufficiently accurate structure is determined) or to improve itsaccuracy. Any art recognized modelling method may be used, including,but not limited to, parameterized models specific to particularbiopolymers such as proteins or nucleic acids, molecular dynamics modelsbased on computing molecular motions, statistical mechanics models basedon thermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. Exemplary forcefields that areknown in the art and can be used in such methods include, but are notlimited to, the Constant Valence Force Field (CVFF), the AMBER forcefield and the CHARM force field. The incomplete or less accurateexperimental structures can serve as constraints on the complete andmore accurate structures computed by these modeling methods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a search can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential target or pathway geneproduct modulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modelling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites oftarget or pathway gene or gene products and related transduction andtranscription factors will be apparent to those of skill in the art.

Examples of molecular modelling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modelling of drugs interactive withspecific proteins, such as Rotivinen et al., 1988, Acta PharmaceuticalFennica 97:159-166; Ripka, (Jun. 16, 1988), New Scientist 54-57;McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122;Perry and Davies, OSAR: Quantitative Structure-Activity Relationships inDrug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989Proc. R. Soc. Lond. 236:125-140 and 1-162; and, with respect to a modelreceptor for nucleic acid components, Askew et al., 1989, J. Am. Chem.Soc. 111:1082-1090. Other computer programs that screen and graphicallydepict chemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

Although generally described above with reference to design andgeneration of compounds which could alter binding, one could also screenlibraries of known compounds, including natural products or syntheticchemicals, and biologically active materials, including proteins, forcompounds which are inhibitors or activators.

Compounds identified via assays such as those described herein can beuseful, for example, for ameliorating the symptoms of immune disorders.For example, in instances in which a TH cell subpopulation-relateddisorder situation results from a lower overall level of 103 geneexpression, 103 gene product, and/or 103 gene product activity in a cellor in tissue involved in such a disorder, compounds that interact withthe 103 gene product can include ones which accentuate or amplify theactivity of the bound 103 gene protein. Such compounds would bring aboutan effective increase in the level of 103 gene activity, thusameliorating symptoms. In instances whereby mutations within the 103gene cause aberrant 103 gene proteins to be made which have adeleterious effect that leads to a TH cell subpopulation-relateddisorder, or, alternatively, in instances whereby normal 103 geneactivity is necessary for a TH cell subpopulation-related disorder tooccur, compounds that bind 103 gene protein can be identified thatinhibit the activity of the bound 103 gene protein. Assays foridentifying additional compounds as well as for testing theeffectiveness of compounds, identified by, for example, techniques, suchas those described in Section 5.4.1-5.4.4, are discussed, below, inSection 5.4.5.

5.4.1. IN VITRO SCREENING ASSAYS FOR COMPOUNDS THAT BIND TO A TARGETGENE PRODUCT

In vitro systems can be designed to identify compounds capable ofbinding the 103 gene products of the invention. Compounds identified canbe useful, for example, in modulating the activity of wild type and/ormutant 103 gene products, can be utilized in screens for identifyingcompounds that disrupt normal 103 gene product interactions, or can inthemselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the103 gene product involves preparing a reaction mixture of a 103 geneproduct and a test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexwhich can be removed and/or detected in the reaction mixture. Theseassays can be conducted in a variety of ways. For example, one method toconduct such an assay involves anchoring 103 gene product or the testsubstance onto a solid phase and detecting 103 gene product/testcompound complexes anchored on the solid phase at the end of thereaction. In one embodiment of such a method, the 103 gene product canbe anchored onto a solid surface, and the test compound, which is notanchored, can be labeled, either directly or indirectly.

In practice, microtiter plates can conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, can be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for the 103 geneproduct or the test compound to anchor any complexes formed in solution,and a labeled antibody specific for the other component of the possiblecomplex to detect anchored complexes.

As an example, and not by way of limitation, techniques such as thosedescribed in this section can be utilized to identify compounds whichbind to the 103 gene product. For example, a 103 gene product can becontacted with a compound for a time sufficient to form a 103 geneproduct/compound complex and then such a complex can be detected.

Alternatively, the compound can be contacted with the 103 gene productin a reaction mixture for a time sufficient to form a 103 geneproduct/compound complex, and then such a complex can be separated fromthe reaction mixture.

Among the 103 gene products which can be utilized for such methods are,for example, rat, murine and human 103 gene products, including, but notlimited to any of the 103 gene products described above in Section 5.2or a naturally occurring variant thereof.

The term “naturally occurring variant,” as used herein refers to anamino acid sequence homologous to the 103 gene product in the same or adifferent species, such as, for example, an allelic variant of the 103gene product which maps to the same chromosomal location as thenucleotide sequences encoding the 103 gene products described above inSection 5.2, or a location syntenic to such a location. Among theallelic variants which can be utilized herein are allelic variantsequences encoded by a nucleotide sequence that hybridizes understringent conditions described, e.g., in Section 5.1 above, to thecomplement of a nucleotide sequence encoding the 103 gene productsdescribed hereinabove.

5.4.2. ASSAYS FOR PROTEINS THAT INTERACT WITH THE 103 GENE PROTEIN

Any method suitable for detecting protein-protein interactions can beemployed for identifying novel 103 protein-cellular or extracellularprotein interactions. Among the traditional methods which can beemployed are co-immunoprecipitation, crosslinking and co-purificationthrough gradients or chromatographic columns. Utilizing procedures suchas these allows for the identification of proteins that interact with a103 gene product. Once identified, such proteins can be used, forexample, to treat or modulate symptoms of an immune disorder, includingan immune disorder associated with a TH2 or TH2-like immune responsesuch as an atopic condition (e.g., asthma or allergy). Once identified,such proteins that interact with a 103 gene product can also be used, inconjunction with standard techniques, to identify the corresponding genethat encodes the protein which interacts with the 103 gene product. Forexample, at least a portion of the amino acid sequence of the geneproduct can be ascertained using techniques well known to those of skillin the art, such as via the Edman degradation technique (see, e.g.,Creighton, 1983, Proteins: Structures and Molecular Principles, W.H.Freeman & Co., N.Y., pp. 34-49). The amino acid sequence obtained can beused as a guide for the generation of oligonucleotide mixtures that canbe used to screen for gene sequences. Screening can be accomplished, forexample, by standard hybridization or PCR techniques. Techniques for thegeneration of oligonucleotide mixtures and for screening are well-known.(See, e.g., Ausubel, supra., and PCR Protocols: A Guide to Methods andApplications, 1990, Innis, M. et al., eds. Academic Press, Inc., NewYork).

Additionally, methods can be employed which result in the simultaneousidentification of genes which encode proteins interacting with a 103gene protein. These methods include, for example, probing expressionlibraries with labeled 103 gene protein, using this protein in a mannersimilar to the well known technique of antibody probing of λgt11libraries.

One method which detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration purposes only and not byway of limitation. One version of this system has been described (Chienet al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:9578-9582) and iscommercially available from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one consists of the DNA-binding domain of atranscription activator protein fused to a known protein, in this case,a 103 gene protein known to be involved in TH cell subpopulationdifferentiation or effector function, or in TH cellsubpopulation-related disorders, and the other consists of the activatorprotein's activation domain fused to an unknown protein that is encodedby a cDNA which has been recombined into this plasmid as part of a cDNAlibrary. The plasmids are transformed into a strain of the yeastSaccharomyces cerevisiae that contains a reporter gene (e.g., lacZ)whose regulatory region contains the transcription activator's bindingsites. Either hybrid protein alone cannot activate transcription of thereporter gene, the DNA-binding domain hybrid cannot because it does notprovide activation function, and the activation domain hybrid cannotbecause it cannot localize to the activator's binding sites. Interactionof the two hybrid proteins reconstitutes the functional activatorprotein and results in expression of the reporter gene, which isdetected by an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screenactivation domain libraries for proteins that interact with a known“bait” gene product. By way of example, and not by way of limitation,103 gene products known to be involved in TH cell subpopulation-relateddisorders and/or differentiation, maintenance, and/or effector functionof the subpopulations can be used as the bait gene products. Totalgenomic or cDNA sequences are fused to the DNA encoding an activationdomain. This library and a plasmid encoding a hybrid of the bait geneproduct fused to the DNA-binding domain are cotransformed into a yeastreporter strain, and the resulting transformants are screened for thosethat express the reporter gene. For example, and not by way oflimitation, the bait (e.g., 103) gene can be cloned into a vector suchthat it is translationally fused to the DNA encoding the DNA-bindingdomain of the GAL4 protein. These colonies are purified and the libraryplasmids responsible for reporter gene expression are isolated. DNAsequencing is then used to identify the proteins encoded by the libraryplasmids.

A cDNA library of the cell line from which proteins that interact withbait (e.g., 103) gene product are to be detected can be made usingmethods routinely practiced in the art. According to the particularsystem described herein, for example, the cDNA fragments can be insertedinto a vector such that they are translationally fused to the activationdomain of GAL4. This library can be co-transformed along with the baitgene-GAL4 fusion plasmid into a yeast strain which contains a lacZ genedriven by a promoter which contains GAL4 activation sequence. A cDNAencoded protein, fused to GAL4 activation domain, that interacts withbait gene product will reconstitute an active GAL4 protein and therebydrive expression of the lacZ gene. Colonies which express lacZ can bedetected by their blue color in the presence of X-gal. The cDNA can thenbe purified from these strains, and used to produce and isolate the baitgene-interacting protein using techniques routinely practiced in theart.

5.4.3. ASSAYS FOR COMPOUNDS THAT INTERFERE WITH 103 GENEPRODUCT/CELLULAR MACROMOLECULE INTERACTION

The 103 gene products of the invention can, in vivo, interact with oneor more cellular or extracellular macromolecules, such as proteins. Suchmacromolecules can include, but are not limited to, nucleic acidmolecules and those proteins identified via methods such as thosedescribed, above, in Section 5.4.2. For purposes of this discussion,such cellular and extracellular macromolecules are referred to herein as“binding partners”. Compounds that disrupt such interactions can beuseful in regulating the activity of a 103 gene protein, especiallymutant 103 gene proteins. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and the like, asdescribed, for example, in Section 5.4.1 above.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between a 103 gene product and itscellular or extracellular binding partner or partners involves preparinga reaction mixture containing the 103 gene product and the bindingpartner under conditions and for a time sufficient to allow the two tointeract and bind, thus forming a complex. In order to test a compoundfor inhibitory activity, the reaction mixture is prepared in thepresence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of 103 gene product and its cellular orextracellular binding Partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the 103 gene protein and the cellular or extracellularbinding partner is then detected. The formation of a complex in thecontrol reaction, but not in the reaction mixture containing the testcompound, indicates that the compound interferes with the interaction ofthe 103 gene protein and the interactive binding partner. Additionally,complex formation within reaction mixtures containing the test compoundand normal 103 gene protein can also be compared to complex formationwithin reaction mixtures containing the test compound and a mutant 103gene protein. This comparison can be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal 103 gene proteins.

The assay for compounds that interfere with the interaction of the 103gene products and binding partners can be conducted in a heterogeneousor homogeneous format. Heterogeneous assays involve anchoring either the103 gene product or the binding partner onto a solid phase and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants can bevaried to obtain different information about the compounds being tested.For example, test compounds that interfere with the interaction betweenthe 103 gene products and the binding partners, e.g., by competition,can be identified by conducting the reaction in the presence of the testsubstance; i.e., by adding the test substance to the reaction mixtureprior to or simultaneously with the 103 gene protein and interactivecellular or extracellular binding partner. Alternatively, test compoundsthat disrupt preformed complexes, e.g. compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

In a heterogeneous assay system, either the 103 gene protein or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtiter plates are convenientlyutilized. The anchored species can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished simplyby coating the solid surface with a solution of the 103 gene product orbinding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored can be used to anchor thespecies to the solid surface. The surfaces can be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the 103 gene protein andthe interactive cellular or extracellular binding partner is prepared inwhich either the 103 gene product or its binding partner is labeled, butthe signal generated by the label is quenched due to complex formation(see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes thisapproach for immunoassays). The addition of a test substance thatcompetes with and displaces one of the species from the preformedcomplex will result in the generation of a signal above background. Inthis way, test substances which disrupt 103 gene protein/cellular orextracellular binding partner interaction can be identified.

In a particular embodiment, the target gene product can be prepared forimmobilization using recombinant DNA techniques described in Section5.2, above. For example, the 103 gene coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector, such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion protein. The interactive cellular or extracellularbinding partner can be purified and used to raise a monoclonal antibody,using methods routinely practiced in the art and described above, inSection 5.3. This antibody can be labeled with the radioactive isotope¹²⁵I, for example, by methods routinely practiced in the art. In aheterogeneous assay, e.g., the GST-103 gene fusion protein can beanchored to glutathione-agarose beads. The interactive cellular orextracellular binding partner can then be added in the presence orabsence of the test compound in a manner that allows interaction andbinding to occur. At the end of the reaction period, unbound materialcan be washed away, and the labeled monoclonal antibody can be added tothe system and allowed to bind to the complexed components. Theinteraction between the 103 gene protein and the interactive cellular orextracellular binding partner can be detected by measuring the amount ofradioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-103 gene fusion protein and the interactivecellular or extracellular binding partner can be mixed together inliquid in the absence of the solid glutathione-agarose beads. The testcompound can be added either during or after the species are allowed tointeract. This mixture can then be added to the glutathione-agarosebeads and unbound material is washed away. Again the extent ofinhibition of the 103 gene product/binding partner interaction can bedetected by adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the 103 gene product and/or the interactive cellular or extracellularbinding partner (in cases where the binding partner is a protein), inplace of one or both of the full length proteins. Any number of methodsroutinely practiced in the art can be used to identify and isolate thebinding sites. These methods include, but are not limited to,mutagenesis of the gene encoding one of the proteins and screening fordisruption of binding in a co-immunoprecipitation assay. Compensatingmutations in the gene encoding the second species in the complex canthen be selected. Sequence analysis of the genes encoding the respectiveproteins will reveal the mutations that correspond to the region of theprotein involved in interactive binding. Alternatively, one protein canbe anchored to a solid surface using methods described in this Sectionabove, and allowed to interact with and bind to its labeled bindingpartner, which has been treated with a proteolytic enzyme, such astrypsin. After washing, a short, labeled peptide comprising the bindingdomain can remain associated with the solid material, which can beisolated and identified by amino acid sequencing. Also, once the genecoding for the cellular or extracellular binding partner is obtained,short gene segments can be engineered to express peptide fragments ofthe protein, which can then be tested for binding activity and purifiedor synthesized.

For example, and not by way of limitation, a 103 gene product can beanchored to a solid material as described, above, in this Section, bymaking a GST-103 gene fusion protein and allowing it to bind toglutathione agarose beads. The interactive cellular or extracellularbinding partner can be labeled with a radioactive isotope, such as ³⁵S,and cleaved with a proteolytic enzyme such as trypsin. Cleavage productscan then be added to the anchored GST-103 gene fusion protein andallowed to bind. After washing away unbound peptides, labeled boundmaterial, representing the cellular or extracellular binding partnerbinding domain, can be eluted, purified, and analyzed for amino acidsequence by well known methods. Peptides so identified can be producedsynthetically or fused to appropriate facilitative proteins using wellknown recombinant DNA technology.

5.4.4. CELL AND ANIMAL-BASED MODEL SYSTEMS

Described herein are cell- and animal-based systems of the presentinvention which act as models for immune disorders and as models of THcell subpopulation differentiation, maintenance, and/or effectorfunction. These systems can be used in a variety of applications. Forexample, such model systems can be used to test compounds identified,e.g., using the in vitro assays described in Section 5.4.1, above, fortheir ability and/or effectiveness in treating (e.g., ameliorating ormodulating symptoms of) immune-related disorders. Thus, the animal- andcell-based models of the invention can be used to identify drugs,pharmaceuticals, therapies and interventions which can be effective intreating immune disorders such as TH cell subpopulation-relateddisorders. In addition, as described in detail, below, in Section 5.7.1,such animal models can be used to determine the LD₅₀ and the ED₅₀ inanimal subjects, and such data can be used to determine the in vivoefficacy of potential immune disorder treatments.

Animal-Based Systems:

Animal-based model systems of TH cell subpopulation-related disorderscan include both non-recombinant animals as well as recombinantlyengineered transgenic animals.

Animal models for TH cell subpopulation-related disorders can include,for example, genetic models. For example, such animal models can includeLeishmania resistance models, experimental allergic encephalomyelitismodels and (BALB/c Cr×DBA/2Cr) F1 mice. These latter mice develop afatal disseminated disease by systemic infection with virulent Candidaalbicans associated with strong TH2-like responses. Additionally, wellknown mouse models for asthma can be utilized to study the ameliorationof symptoms caused in immune disorders, such as allergy and asthma, thatare associated with a strong TH2 or TH2-like response. (See, forexample, N. W. Lukacs et al., 1994, Am. J. Resp. Cell Mol. Biol.10:526-532; S. H. Gavett et al. al., 1994, Am. J. Resp. Cell Mol. Biol.10:587-593.) Further, the animal model, murine acquired immunodeficiencysyndrome (MAIDS; B. Kanagawa et al., 1993, Science 262:240; M. Makino etal., 1990, J. Imm. 144:4347) can be used for such studies.

Alternatively, such well known animal models as SCIDhu mice (see forexample, H. Kaneshima et al., 1994, Curr. Opin. Imm. 6:327-333) whichrepresents an in vivo model of the human hematolymphoid system, can beutilized. Further, the RAG-2-deficient blastocyst complementationtechnique (J. Chen et al., 1993, Proc. Natl. Acad. Sci. USA90:4528-4532; Y. Shinkai et al., 1992, Cell 68:855-867) can be utilizedto produce mice containing, for example, humanized lymphocytes and/orwhich express target gene sequences. Still further, targeting techniquesdirected specifically to T cells, for example, the technique of Gu etal. (1994, Science 265:103-106) can be utilized to produce animalscontaining transgenes in only T cell populations.

Further, animal models such as the adoptive transfer model described,e.g., in L. Cohn et al., 1997, J. Exp. Med. 186:1737-1747) and describedand utilized in Section 6.4, below, can be used. In such an animalsystem, aeroallergen provocation of TH1 or TH2 recipient mice results inTH effector cell migration to the airways and is associated with anintense neutrophilic (TH1) and eosinophilic (TH2) lung mucosalinflammatory response. The animal model represents an accepted model forasthma.

Animal models exhibiting TH cell subpopulation-related disorder-likesymptoms can be engineered by utilizing, for example, target genesequences such as the 103 gene sequences described, above, in Section5.1, in conjunction with techniques for producing transgenic animalsthat are well known to those of skill in the art. For example, targetgene sequences can be introduced into, and overexpressed and/ormisexpressed in, the genome of the animal of interest, or, if endogenoustarget gene sequences are present, they can either be overexpressed,misexpressed, or, alternatively, can be disrupted in order tounderexpress or inactivate target gene expression. The construction andcharacterization of exemplary 103 gene transgenic animals is describedin Section 6.3, below.

In order to overexpress or misexpress a target gene sequence (e.g., a103 gene sequence), the coding portion of the target gene sequence canbe ligated to a regulatory sequence which is capable of driving highlevel gene expression or expression in a cell type in which the gene isnot normally expressed in the animal and/or cell type of interest. Suchregulatory regions will be well known to those of skill in the art, andcan be utilized in the absence of undue experimentation.

For underexpression of an endogenous target gene sequence (e.g., of anendogenous 103 gene sequence), such a sequence can be isolated andengineered such that when reintroduced into the genome of the animal ofinterest, the endogenous target gene alleles will be inactivated.Preferably, the engineered target gene sequence is introduced via genetargeting such that the endogenous target sequence is disrupted uponintegration of the engineered target gene sequence into the animal'sgenome. Gene targeting is discussed, below, in this Section.

Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, squirrels, monkeys, and chimpanzees can be used togenerate animal models of TH cell subpopulation-related disorders.

Any technique known in the art can be used to introduce a target genetransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol. Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, Intl. Rev.Cytol. 115:171-229, which is incorporated by reference herein in itsentirety.

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. (See,for example, techniques described by Jakobovits, 1994, Curr. Biol.4:761-763.) The transgene can be integrated as a single transgene or inconcatamers, e.g., head-to-head tandems or head-to-tail tandems. Thetransgene can also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal. (Lasko et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236). Theregulatory sequences required for such a cell-type specific activationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

When it is desired that the target gene transgene be integrated into thechromosomal site of the endogenous target gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous targetgene of interest are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of, the nucleotide sequence of the endogenous target gene.The transgene can also be selectively introduced into a particular celltype, thus inactivating the endogenous gene of interest in only thatcell type, by following, for example, the teaching of Gu et al. (Gu etal., 1994, Science 265:103-106). The regulatory sequences required forsuch a cell-type specific inactivation will depend upon the particularcell type of interest, and will be apparent to those of skill in theart.

Once transgenic animals have been generated, the expression of therecombinant target gene and protein can be assayed utilizing standardtechniques. Initial screening can be accomplished by Southern blotanalysis or PCR techniques to analyze animal tissues and to assaywhether integration of the transgene has taken place. The level of mRNAexpression of the transgene in the tissues of the transgenic animals canalso be assessed using techniques which include but are not limited toNorthern blot analysis of tissue samples obtained from the animal, insitu hybridization analysis, and RT-PCR. Samples of targetgene-expressing tissue, can also be evaluated immunocytochemically usingantibodies specific for the target gene transgene gene product ofinterest.

103 gene transgenic animals that express 103 gene mRNA or 103 genetransgene peptide (detected immunocytochemically, using antibodiesdirected against target gene product epitopes) at easily detectablelevels can be further evaluated to identify those animals which displaycharacteristic TH cell subpopulation-related disorder-like symptoms, orexhibit characteristic TH cell subpopulation differentiation phenotypes.TH1-like-related disorder symptoms can include, for example, thoseassociated with chronic inflammatory diseases and disorders, such asCrohn's disease, reactive arthritis (including but not limited to Lymedisease), insulin-dependent diabetes, organ-specific autoimmunity,(including but not limited to multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease), contact dermatitis, psoriasis, graftrejection, graft versus host disease and sarcoidosis to name a few.TH2-like-related disorder symptoms can include, but are not limited to,those associated with atopic conditions such as asthma and allergy suchas allergic rhinitis, gastrointestinal allergies, (including but notlimited to food allergies); eosinophilia; conjunctivitis; glomerularnephritis; certain pathogen susceptibilities such as helminthic (e.g.,leishmaniasis); and certain viral infections, including HIV, andbacterial infections (for example, tuberculosis and lepromatousleprosy).

Additionally, specific cell types within the transgenic animals can beanalyzed and assayed for cellular phenotypes characteristic of TH cellsubpopulation-related disorders. Such cellular phenotypes can include,for example, differential cytokine expression characteristic of the THcell subpopulation of interest. Further, such cellular phenotypes caninclude an assessment of a particular cell type's fingerprint pattern ofexpression and its comparison to known fingerprint expression profilesof the particular cell type in animals exhibiting specific TH cellsubpopulation-related disorders. Such transgenic animals serve assuitable model systems for TH cell-related disorders.

Once target gene transgenic founder animals are produced (i.e., thoseanimals which express target gene products (e.g., 103 gene proteins) incells or tissues of interest, and which, preferably, exhibit symptoms ofTH cell subpopulation-related disorders), they can be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound target gene transgenics that express the target genetransgene of interest at higher levels because of the effects ofadditive expression of each target gene transgene; crossing ofheterozygous transgenic animals to produce animals homozygous for agiven integration site in order to both augment expression and eliminatethe possible need for screening of animals by DNA analysis; crossing ofSeparate homozygous lines to produce compound heterozygous or homozygouslines; breeding animals to different inbred genetic backgrounds so as toexamine effects of modifying alleles on expression of the target genetransgene and the development of TH cell subpopulation-relateddisorder-like symptoms. One such approach is to cross the target genetransgenic founder animals with a wild type strain to produce an F1generation that exhibits TH cell subpopulation-related disorder-likesymptoms, such as those described above. The F1 generation can then beinbred in order to develop a homozygous line, if it is found thathomozygous target gene transgenic animals are viable.

Cell-Based Assays:

Cells that contain and express 103 gene sequences which encode 103 geneprotein, and, further, exhibit cellular phenotypes associated with a THcell subpopulation and/or a TH cell subpopulation-related disorder ofinterest, can be utilized to identify compounds that exhibit and/or canbe tested for an ability to ameliorate TH cell subpopulation-relateddisorder symptoms. Cellular phenotypes which can indicate an ability toameliorate TH cell subpopulation-related disorder symptoms can include,for example, an inhibition or potentiation of cytokine or cell surfacemarker expression associated with the TH cell subpopulation of interest,or, alternatively, an inhibition or potentiation of specific TH cellsubpopulations.

Further, the fingerprint pattern of gene expression of cells of interestcan be analyzed and compared to the normal, non-TH cellsubpopulation-related disorder fingerprint pattern. Those compoundswhich cause cells exhibiting TH cell subpopulation-related disorder-likecellular phenotypes to produce a fingerprint pattern more closelyresembling a normal fingerprint pattern for the cell of interest can beconsidered candidates for further testing regarding an ability toameliorate TH cell subpopulation-related disorder symptoms.

Cells which can be utilized for such assays can, for example, includenon-recombinant cell lines, such as Dorris, AE7, D10.G4, DAX, D1.1 andCDC25 cell lines. In addition, purified primary naive T cells derivedfrom either transgenic or non-transgenic strains can also be used.

Further, cells which can be used for such assays can also includerecombinant, transgenic cell lines. For example, the TH cellsubpopulation-related disorder animal models of the invention,discussed, above, in Section 5.7.1, can be used to generate, forexample, TH1-like and/or TH2-like cell lines that can be used as cellculture models for the disorder of interest. While primary culturesderived from TH cell subpopulation-related disorder transgenic animalscan be utilized, the generation of continuous cell lines is preferred.For examples of techniques which can be used to derive a continuous cellline from the transgenic animals, see, e.g., Small et al., 1985, Mol.Cell. Biol. 5:642-648.

Alternatively, cells of a cell type known to be involved in TH cellsubpopulation-related disorders can be transfected with sequencescapable of increasing or decreasing the amount of 103 gene expressionwithin the cell. For example, 103 gene sequences can be introduced into,and overexpressed in, the genome of the cell of interest, or, ifendogenous 103 gene sequences are present, they can either beoverexpressed or, alternatively, can be disrupted in order tounderexpress or inactivate target gene expression.

In order to overexpress a 103 gene sequence, the coding portion of the103 gene sequence can be ligated to a regulatory sequence which iscapable of driving gene expression in the cell type of interest. Suchregulatory regions will be well known to those of skill in the art, andcan be utilized in the absence of undue experimentation.

For underexpression of an endogenous 103 gene sequence, such a sequencecan be isolated and engineered such that when reintroduced into thegenome of the cell type of interest, the endogenous 103 gene alleleswill be inactivated. Preferably, the engineered 103 gene sequence isintroduced via gene targeting such that the endogenous 103 sequence isdisrupted upon integration of the engineered 103 gene sequence into thecell's genome. Gene targeting is discussed above.

Transfection of 103 gene sequence nucleic acid can be accomplished byutilizing standard techniques. See, for example, Ausubel, 1989, supra.Transfected cells should be evaluated for the presence of therecombinant target gene sequences, for expression and accumulation oftarget gene mRNA, and for the presence of recombinant target geneprotein production. In instances wherein a decrease in target geneexpression is desired, standard techniques can be used to demonstratewhether a decrease in endogenous target gene expression and/or in targetgene product production is achieved.

Cells to be utilized can, for example, be stimulated or activated as,described e.g., in the Examples presented below.

5.4.5. ASSAYS FOR AMELIORATION OF IMMUNE DISORDER SYMPTOMS AND/OR THEMODULATION OF 103 GENE PRODUCT FUNCTION

Any of the binding compounds, including but not limited to, compoundssuch as those identified in the foregoing assay systems, can be testedfor the ability to ameliorate symptoms of immune disorders, including,for example, any of the TH cell subpopulation-related disordersdescribed herein. Cell-based and animal model-based assays for theidentification of compounds exhibiting such an ability to ameliorateimmune disorder symptoms are described below. Exemplary embodiments ofcell-based and animal-model assays which can be used in the methods andcompositions of the present invention are further described in theexamples presented in Sections 6.4 and 6.6, below. In particular, anexemplary cell-based assay is presented in Section 6.6. An assay usingan exemplary and art recognized animal model for asthma is alsodescribed, below, in Section 6.4.

First, cell-based systems such as those described, above, in Section5.4.4, can be used to identify compounds which can act to ameliorate THcell subpopulation-related disorder symptoms. For example, such cellsystems can be exposed to a compound, suspected of exhibiting an abilityto ameliorate the disorder symptoms, at a sufficient concentration andfor a time sufficient to elicit such an amelioration in the exposedcells. After exposure, the cells are examined to determine whether oneor more of the TH cell subpopulation-related disorder-like cellularphenotypes has been altered to resemble a phenotype more likely toproduce a lower incidence or severity of disorder symptoms.

Taking, as a non-limiting example, the TH cell subpopulation-relateddisorders of allergy and asthma, which are, specifically,TH2-like-related disorders (e.g., the disorders are associated with astrong TH2 or TH2-like immune response), any TH2 or TH2-like cell systemcan be utilized. Upon exposure to such cell systems, compounds can beassayed for their ability to modulate the TH2-like phenotype of suchcells, such that the cells exhibit loss of a TH2-like phenotype.Compounds with such TH2 modulatory capability represent ones which canpotentially exhibit the ability to ameliorate allergy and/orasthma-related symptoms in vivo. The Example presented in Section 6.4,below, describes the successful utilization of a 103 gene product/Igfusion protein, as well as the successful use of a monoclonal antibodydirected against the extracellular domain of the 103 gene product toameliorate symptoms of asthma in an accepted animal model of asthma.

In addition, animal-based systems, such as those described, above, inSection 5.4.4, can be used to identify compounds capable of amelioratingTH cell subpopulation-related disorder-like symptoms. Such animal modelscan be used as test substrates for the identification of drugs,pharmaceuticals, therapies, and interventions which can be effective intreating such disorders. For example, animal models can be exposed to acompound, suspected of exhibiting an ability to ameliorate TH cellsubpopulation-related disorder symptoms, at a sufficient concentrationand for a time sufficient to elicit such an amelioration of the symptomsin the exposed animals. The response of the animals to the exposure, andthus the efficacy of the compound in question, can be monitored byassessing the reversal of disorders associated with TH cellsubpopulation-related disorders of interest. With regard tointervention, any treatments which reverse any aspect of TH cellsubpopulation-related disorder-like symptoms should be considered ascandidates for corresponding human TH cell subpopulation-relateddisorder therapeutic intervention. Dosages of test agents can bedetermined by deriving dose-response curves, as discussed in Section5.7, below.

Gene expression patterns can be utilized in conjunction with eithercell-based or animal-based systems, to assess the ability of a compoundto ameliorate TH cell subpopulation-related disorder-like symptoms. Forexample, the expression pattern of one or more “fingerprint” genes(including, for example, the expression pattern of the 103 gene) canform part of a fingerprint profile which can be then be used in such anassessment. Fingerprint profiles are described, below, in Section 5.8.Fingerprint profiles can be characterized for known states, either THcell subpopulation-related disorder states, or normal TH celldifferentiative states, within the cell- and/or animal-based modelsystems.

5.5. COMPOSITIONS AND METHODS FOR TREATMENT OF IMMUNE DISORDERS AND FORMODULATION OF TH CELL RESPONSIVENESS

Described below are methods and compositions which can be used toameliorate immune disorder symptoms via, for example, a modulation ofthe TH cell subpopulation of interest. The methods and compositionsdescribed herein can also be used to ameliorate immune disorderssymptoms via a modulation of other cell populations, e.g., mast cellpopulations, that specifically express the 103 gene. Such modulation canbe of a positive or negative nature, depending on the specific situationinvolved, but each modulatory event yields a net result in whichsymptoms of the immune disorder are ameliorated. Further, describedbelow are methods for the modulation of TH cell responsiveness toantigen.

It is possible that a TH cell subpopulation-related disorder or otherimmune disorder, can occur as a result of normal 103 gene activityduring the course of, for example, exposure to a certain antigen whichelicits an immune response that leads to the development of thedisorder. For example, the disorders of asthma and allergy are likelycandidates of disorders having such a mechanism. Additionally, adisorder can be brought about, at least in part, by an abnormally highlevel of 103 gene product, or by the presence of a 103 gene productexhibiting an abnormal activity. As such, a technique which elicits anegative modulatory effect, i.e., brings about a reduction in the leveland/or activity of 103 gene product, or alternatively, brings about adepletion of the TH cell subpopulation (such as a TH2 cellsubpopulation, e.g., via a physical reduction in the number of cellsbelonging to the TH cell subpopulation), would effect an amelioration ofTH cell subpopulation-related disorder symptoms in either of the abovescenarios.

Negative modulatory techniques for the reduction of gene expressionlevels or gene product activity levels, (including 103 gene expressionlevels or 103 gene product activity levels, either normal or abnormal),and for the reduction in the number of specific TH cell subpopulationcells are discussed in Section 5.6.1, below.

Alternatively, it is possible that a TH cell subpopulation-relateddisorder or other immune disorders can be brought about, at least inpart, by the absence or reduction of the level of 103 gene expression, areduction in the level of a 103 gene product's activity, or a reductionin the overall number of cells belonging to a specific TH cellsubpopulation (e.g., of a TH2 cell subpopulation). As such, a techniquewhich elicits a positive modulatory effect, i.e., brings about anincrease in the level of 103 gene expression and/or the activity of suchgene products, or, alternatively, a stimulation of the TH cellsubpopulation (e.g., via a physical increase in the number of cellsbelonging to a TH cell subpopulation such as a TH2 cell subpopulation),would effect an amelioration of immune disorder symptoms.

For example, a reduction in the overall number of TH1-like cellsrelative to TH2-like cells within a HIV-infected individual cancorrelate with the progression to AIDS (Clerci et al., 1993, J. Clin.Invest. 91:759; Clerci. et al., 1993, Science 262:1721; Maggi et al.,1994, Science 265:244). A treatment capable of increasing the number ofTH1-like cells relative to TH2-like cells within an HIV-infectedindividual may, therefore, serve to prevent or slow the progression todisease.

Positive modulatory techniques for increasing gene expression levels orgene product activity levels, including 103 gene expression levels and103 gene product activity levels, and for increasing the level ofspecific TH cell subpopulation cells are discussed, below, in Section5.6.2.

Among the immune disorders whose symptoms can be ameliorated are TH1 orTH1-like related immune disorders (i.e., immune disorders associatedwith a strong or inappropriate TH1 or TH1-like immune response) and TH2or TH2-like related immune disorders (i.e., immune disorders associatedwith a strong or inappropriate TH2 or TH2-like immune response).Examples of TH1 or TH1-like related disorders include chronicinflammatory diseases and disorders such as Crohn's disease, reactivearthritis including Lyme disease, insulin-dependent diabetes,organ-specific autoimmunity (including multiple sclerosis, Hashimoto'sthyroiditis and Grave's disease), contact dermatitis, psoriasis, graftrejection, graft versus host disease and sarcoidosis. Examples of TH2 orTH2-like related disorders include atopic conditions, such as asthma andallergy (including allergic rhinitis), gastrointestinal allergiesincluding food allergies, eosinophilia, conjunctivitis, glomerularnephritis, systemic lupus erythematosus, scleroderma, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis), certain viralinfections, including HIV, and bacterial infections such as tuberculosisand lepromatous leprosy.

The methods described herein can additionally be utilized the modulatethe level of responsiveness, for example, responsiveness to antigen, ofa TH cell subpopulation. Such methods are important in that many immunedisorders involve inappropriate rather than insufficient immuneresponses. For example, disorders such as atopic, IgE-mediated allergicconditions, including asthma, pathogen susceptibilities and chronicinflammatory disease, involve strong but counterproductive TH2-mediatedimmune responses. Further, inappropriate TH1-mediated immune responsesto self-antigens is central to the development of such disorders asmultiple sclerosis, psoriasis, insulin dependent diabetes, Hashimoto'sthyroiditis and Crohn's disease.

Methods for modulating TH cell responsiveness can comprise, for example,contacting a compound to a TH cell so that the responsiveness of the Thelper cell is modulated relative to the responsiveness of the T helpercell in the absence of the compound. The modulation can increase ordecrease the responsiveness of the TH cell. Any of the techniquesdescribed, below, in Sections 5.6.1-5.6.3 can be utilized to effect anappropriate modulation of TH cell responsiveness.

The methods described herein can additionally be utilized to modulatethe other cell populations, such as mast cell populations, thatspecifically express the 103 gene product. In particular, many immunedisorders, including, the immune disorders described above and, inparticular, atopic conditions such as asthma and allergy, are mediatedby mast cell populations as well as by other populations of immune cellssuch as TH1, TH1-like, TH2 or TH2-like subpopulations. Thus, the methodsand compositions described herein can also be used to treat immunedisorders, including atopic conditions such as asthma and allergy, bytargeting other cell populations (e.g., mast cells) that are involved insuch immune disorders, in addition to or even instead of TH cellsubpopulations.

The 103 gene is also expressed in human mast cells, as demonstrated inthe Example presented in Section 6.5, below. Thus, the above-describedcompositions (e.g., natural ligands, derivatives of natural ligands andantibodies that specifically bind to the 103 gene product) can also beutilized to modulate the number of mast cells present and/or to modulatethe amount of mast cell activity or mast cell cytokine production (e.g.,from the degranulation of mast cells). Thus conditions, including atopicconditions such as asthma and allergy, that involve or are mediated bymast cell activity (often in addition to TH2 or TH2-like activity) canbe treated by using the methods and compositions of the invention totarget mast cells and/or mast cell activity as well as (or instead of)TH2 cells and/or TH2 cell activity.

5.5.1. NEGATIVE MODULATORY TECHNIQUES

As discussed, above, successful treatment of certain immune disorderscan be brought about by techniques which serve to inhibit the expressionor activity of 103 gene products, or which, alternatively, serve toreduce the overall number of cells belonging to a specific TH cellsubpopulation (e.g., a TH2 cell subpopulation).

For example, compounds such as those identified through assaysdescribed, above, in Section 5.4, which exhibit negative modulatoryactivity, can be used in accordance with the invention to amelioratesymptoms of certain immune disorders. As discussed in Section 5.4,above, such molecules can include, but are not limited to peptides (suchas, for example, peptides representing soluble extracellular portions ofa 103 gene product), phosphopeptides, small organic or inorganicmolecules, or antibodies (including, for example, polyclonal,monoclonal, human, humanized, anti-idiotypic, chimeric or single chainantibodies, and FAb, F(ab′)₂ and FAb expression library fragments, andepitope-binding fragments thereof). In one embodiment, for example,antibodies directed against a 103 gene product, preferably anextracellular or extracellular portion of a 103 gene product, can beutilized. Techniques for the determination of effective doses andadministration of such compounds are described, below, in Section 5.7.1.

Antisense and ribozyme molecules which inhibit expression of the 103gene can also be used in accordance with the invention to reduce thelevel of 103 gene expression, thus effectively reducing the level oftarget gene activity. Still further, triple helix molecules can beutilized in reducing the level of 103 gene activity. Such techniques aredescribed hereinbelow.

Techniques for the depletion of specific TH cell subpopulations arediscussed, hereinbelow, in Section 5.5.3. Such techniques can takeadvantage of, for example, novel cell surface markers, including the 103gene product, which are specific to the TH cell subpopulation to bedepleted, and can include in vivo or in vitro targeted destruction, or,alternatively, selective purification away, of the TH cell subpopulationof interest.

As discussed above, the 103 gene represents a TH2-specific gene in that103 gene expression is found to be absent TH1 cells as well as all othertissues tested. Further, at least one of the proteins produced by the103 gene is a transmembrane protein. The 103 gene and its products can,therefore, be utilized in the treatment of certain immune disorders suchTH2 cell subpopulation-related disorders. For example, a 103 geneproduct or portions thereof can be utilized, either directly orindirectly, to ameliorate conditions involving inappropriate IgE immuneresponses, including, but not limited to the symptoms which accompanyatopic conditions such as allergy and/or asthma. IgE-type antibodies areproduced by stimulated B cells which require, at least in part, IL-4produced by the TH2 cell subpopulation. Therefore, any treatment,including, for example, the use of a gene 103 product or portionthereof, which reduces the effective concentration of secreted IL-4,e.g., by reducing the number or activity of TH2 cells, can bring about areduction in the level of circulating IgE, leading, in turn, to theamelioration of the conditions stemming from an inappropriate IgE immuneresponse.

There exist a variety of ways in which the TH2 specific 103 geneproducts can be used to effect such a reduction in the activity and/oreffective concentration of TH2 cells.

For example, natural ligands, derivatives of natural ligands andantibodies which bind to the 103 gene product can be utilized to reducethe number of TH2 cells present by either physically separating suchcells away from other cells in a population, thereby deleting the TH2cell subpopulation, or, alternatively, by targeting the specificdestruction of TH2 cells. Such techniques are discussed, below, inSection 5.6.3. Further, such compounds can be used to inhibit theproliferation of TH2 cells.

Additionally, compounds such as 103 gene sequences or gene products canbe utilized to reduce the level of TH2 cell activity, cause a reductionin the production of TH2 associated cytokines such as IL-4, IL-5, IL-10and IL-13, and, ultimately, bring about the amelioration of IgE relateddisorders.

For example, compounds can be administered which compete with endogenousligand for the 103 gene product, e.g., by binding to a ligand-bindingdomain of a 103 gene product. The resulting reduction in the amount ofligand-bound 103 gene transmembrane protein will modulate TH2 cellularactivity. Compounds which can be particularly useful for this purposeinclude, for example, soluble proteins or peptides, such as peptidescomprising the extracellular domain, or portions and/or analogs thereof,of the gene 103 product, including, for example, soluble fusion proteinssuch as Ig-tailed fusion proteins. (For a discussion of the productionof Ig-tailed fusion proteins see, for example, U.S. Pat. No. 5,116,964.)

Production of a 103 gene product/Ig fusion is described in Section 6.2,below. Further, use of a 103 gene product/Ig fusion to successfullyameliorate symptoms in an accepted animal model for asthma is describedin Section 6.4, below.

Among the compounds which can exhibit the ability to ameliorate TH cellsubpopulation-related disorder symptoms are antisense, ribozyme, andtriple helix molecules. Such molecules can be designed to reduce orinhibit either wild type, or if appropriate, mutant 103 gene activity.Techniques for the production and use of such molecules are well knownto those of skill in the art.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to 103 gene mRNA. The antisenseoligonucleotides will bind to the complementary 103 gene mRNAtranscripts and prevent translation. Absolute complementarity, althoughpreferred, is not required. A sequence “complementary” to a portion ofan RNA, as referred to herein, means a sequence having sufficientcomplementarity to be able to hybridize with the RNA, forming a stableduplex; in the case of double-stranded antisense nucleic acids, a singlestrand of the duplex DNA may thus be tested, or triplex formation may beassayed. The ability to hybridize will depend on both the degree ofcomplementarity and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches withan RNA it may contain and still form a stable duplex (or triplex, as thecase may be). One skilled in the art can ascertain a tolerable degree ofmismatch by use of standard procedures to determine the melting point ofthe hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently been shown to be effective atinhibiting translation of mRNAs as well. See generally, Wagner, R.,1994, Nature 372:333-335. Thus, oligonucleotides complementary to eitherthe 5′- or 3′-non-translated, non-coding regions of 103 genes could beused in an antisense approach to inhibit translation of endogenous 103gene mRNA. Oligonucleotides complementary to the 5′ untranslated regionof the mRNA should include the complement of the AUG start codon.Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could be used in accordance withthe invention. Whether designed to hybridize to the 5′-, 3′- or codingregion of 103 gene mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides or at least 50 nucleotides.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the 103 RNA orprotein with that of an internal control RNA or protein. Additionally,it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

The antisense molecules should be delivered to cells which express the103 gene in vivo. A number of methods have been developed for deliveringantisense DNA or RNA to cells; e.g., antisense molecules can be injecteddirectly into the tissue site, or modified antisense molecules, designedto target the desired cells (e.g., antisense linked to peptides orantibodies that specifically bind receptors or antigens expressed on thetarget cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous 103 gene transcripts andthereby prevent translation of the 103 gene mRNA. For example, a vectorcan be introduced in vivo such that it is taken up by a cell and directsthe transcription of an antisense RNA. Such a vector can remain episomalor become chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid, viral, or others known in the art, used for replication andexpression in mammalian cells. Expression of the sequence encoding theantisense RNA can be by any promoter known in the art to act inmammalian, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidinekinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.78:1441-1445), the regulatory sequences of the metallothionein gene(Brinster et al., 1982, Nature 296:39-42), etc. Any type of plasmid,cosmid, YAC or viral vector can be used to prepare the recombinant DNAconstruct which can be introduced directly into the tissue site.Alternatively, viral vectors can be used which selectively infect thedesired tissue.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA (For a review see, for example Rossi, J., 1994, CurrentBiology 4:469-471). The mechanism of ribozyme action involves sequencespecific hybridization of the ribozyme molecule to complementary targetRNA, followed by a endonucleolytic cleavage. The composition of ribozymemolecules must include one or more sequences complementary to the 103gene mRNA, and must include the well known catalytic sequenceresponsible for mRNA cleavage. For this sequence, see U.S. Pat. No.5,093,246, which is incorporated by reference herein in its entirety. Assuch, within the scope of the invention are engineered hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of RNA sequences encoding 103 gene proteins.Ribozyme molecules designed to catalytically cleave 103 gene mRNAtranscripts can also be used to prevent translation of 103 gene mRNA andexpression of 103 gene. (See, e.g., PCT International PublicationWO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). While ribozymes that cleave mRNA at site specificrecognition sequences can be used to destroy 103 gene mRNAs, the use ofhammerhead ribozymes is preferred.

Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, 1988, Nature, 334:585-591. Preferably the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the 103 gene mRNA; i.e., to increase efficiency and minimizethe intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug andCech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature,324:429-433; published International patent application No. WO 88/04300by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216). TheCech-type ribozymes have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. The invention therefore encompasses those Cech-type ribozymeswhich target eight base-pair active site sequences that are present inthe 103 gene.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the 103 gene in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous 103 gene messages andinhibit translation. Because ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

In instances wherein the antisense, ribozyme, and/or triple helixmolecules described herein are utilized to inhibit mutant geneexpression, it is possible that the technique can also efficientlyreduces or inhibits the transcription (triple helix) and/or translation(antisense, ribozyme) of mRNA produced by normal 103 gene alleles. Thus,the possibility can arise wherein the concentration of normal targetgene product present can be lower than is necessary for a normalphenotype. To ensure that substantially normal levels of target geneactivity are maintained in such cases, nucleic acid molecules thatencode and express target gene polypeptides exhibiting normal targetgene activity can be introduced into cells via gene therapy methods,such as those described in Section 5.6.2 below; that do not containsequences susceptible to whatever antisense, ribozyme, or triple helixtreatments are being utilized. Alternatively, in instances whereby thetarget gene encodes an extracellular protein, it can be preferable tocoadminister normal target gene protein in order to maintain therequisite level of target gene activity.

Anti-sense RNA and DNA, ribozyme, and triple helix molecules of theinvention can be prepared by any method known in the art for thesynthesis of DNA and RNA molecules. These include techniques forchemically synthesizing oligodeoxyribonucleotides andoligoribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors which incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.

Endogenous target and/or pathway gene expression can also be reduced byinactivating or “knocking out” the endogenous 103 gene or its promoterusing targeted homologous recombination. (e.g., see Smithies et al.,1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51:503-512;Thompson et al., 1989 Cell 5:313-321; each of which is incorporated byreference herein in its entirety). For example, a mutant, non-functional103 gene (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous 103 gene (either the coding regions orregulatory regions of the 103 gene) can be used, with or without aselectable marker and/or a negative selectable marker, to transfectcells that express the 103 gene in vivo. Insertion of the DNA construct,via targeted homologous recombination, results in inactivation of the103 gene. Such approaches are particularly suited in the agriculturalfield where modifications to ES (embryonic stem) cells can be used togenerate animal offspring with an inactive 103 gene (e.g., see Thomas &Capecchi 1987 and Thompson 1989, supra). Such techniques can also beutilized to generate T cell subpopulation-related disorder animalmodels. It should be noted that this approach can be adapted for use inhumans provided the recombinant DNA constructs are directly administeredor targeted to the required site in vivo using appropriate viralvectors, e.g., herpes virus vectors.

Alternatively, endogenous 103 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the 103 gene (i.e., the 103 gene promoter and/or enhancers) toform triple helical structures that prevent transcription of the 103gene in target cells in the body. (See generally, Helene, C. 1991,Anticancer Drug Des., 6(6):569-84; Helene, C., et al., 1992, Ann, N.Y.Accad. Sci., 660:27-36; and Maher, L. J., 1992, Bioassays14(12):807-15). In yet another embodiment of the invention, the activityof 103 gene can be reduced using a “dominant negative” approach. To thisend, constructs which encode defective 103 gene products can be used ingene therapy approaches to diminish the activity of the 103 gene productin appropriate target cells.

5.5.2. POSITIVE MODULATORY TECHNIQUES

As discussed above, successful treatment of certain immune disorders canbe brought about by techniques which serve to increase the level of 103gene expression or to increase the activity of a 103 gene product, orwhich, or alternatively, serve to effectively increase the overallnumber of cells belonging to a specific TH cell subpopulation (e.g., aTH2 cell subpopulation).

For example, compounds such as those identified through assaysdescribed, above, in Section 5.4, which exhibit positive modulatoryactivity can be used in accordance with the invention to amelioratecertain TH cell subpopulation-related disorder symptoms. As discussed inSection 5.4, above, such molecules can include, but are not limited toproteins or protein fragments of a 103 gene product, including fragmentscorresponding to one or more domains of the target gene product (e.g.,an extracellular domain, an Ig-like domain, a transmembrane domain, or acytosolic domain) or portions thereof. Such molecules can also includepeptides representing soluble extracellular portions of 103 gene producttransmembrane proteins, phosphopeptides, small organic or inorganicmolecules, or antibodies (including, for example, polyclonal,monoclonal, human, humanized, anti-idiotypic, chimeric or single chainantibodies, and FAb, F(ab′)₂ and FAb expression library fragments, andepitope-binding fragments thereof).

For example, a compound, such as a 103 gene protein, can, at a levelsufficient to ameliorate immune disorder symptoms, be administered to apatient exhibiting such symptoms. Any of the techniques discussed,below, in Section 5.7, can be utilized for such administration. One ofskill in the art will readily know how to determine the concentration ofeffective, non-toxic doses of the compound, utilizing techniques such asthose described, below, in Section 5.7.1.

In another embodiment, fragments or peptides representing a functionaldomain of a 103 gene product are administered to an individual atsufficient dosages and such that the fragments or peptides may enhancethe target gene product's activity in the individual, e.g., by mimickingthe function of the target gene product in vivo.

The proteins and peptides which may be used in such methods includesynthetic (e.g., recombinant or chemically synthesized) proteins andpeptides, as well as naturally occurring proteins and peptides. Theproteins and peptides may have both naturally occurring and/ornon-naturally occurring amino acid residues (e.g., D-amino acidresidues) and/or one or more non-peptide bonds (e.g., imino, ester,hydrazide, semicarbazide, and azo bonds). The proteins or peptides mayalso contain additional chemical groups (e.g., functional groups)present at the amino and/or carboxy termini, such that, for example, thestability, bioavailability, and/or inhibitory activity of the peptide isenhanced. Exemplary functional groups include hydrophobic groups (e.g.,carbobenzoxyl, dansyl, and t-butyloxycarbonyl groups) an acetyl group, a9-fluorenylmethoxy-carbonyl group, and macromolecular carrier groups(e.g., lipid-fatty acid conjugates, polyethylene glycol, orcarbohydrates) including peptide groups. Suitable dosages andformulations for administration of such peptides and proteins are alsowell known to those of skill in the art, and are described in Section5.7 above.

In instances wherein the compound to be administered is a peptidecompound, DNA sequences encoding the peptide compound can be directlyadministered to a patient exhibiting immune disorder symptoms, at aconcentration sufficient to produce a level of peptide compoundsufficient to ameliorate the disorder symptoms. Any of the techniquesdiscussed, below, in Section 5.7, which achieve intracellularadministration of compounds, such as, for example, liposomeadministration, can be utilized for the administration of such DNAmolecules. The DNA molecules can be produced, for example, by well knownrecombinant techniques.

In the case of peptides compounds which act extracellularly, the DNAmolecules encoding such peptides can be taken up and expressed by anycell type, so long as a sufficient circulating concentration of peptideresults for the elicitation of a reduction in the immune disordersymptoms. In the case of compounds which act intracellularly, the DNAmolecules encoding such peptides must be taken up and expressed by theTH cell subpopulation of interest at a sufficient level to bring aboutthe reduction of immune disorders.

Any technique which serves to selectively administer DNA molecules tothe TH cell subpopulation of interest is, therefore, preferred, for theDNA molecules encoding intracellularly acting peptides. In the case ofasthma, for example, techniques for the selective administration of themolecules to TH cell subpopulations residing within lung tissue arepreferred.

In instances wherein the TH cell subpopulation-related disorder involvesan aberrant 103 gene, patients can be treated by gene replacementtherapy. One or more copies of a normal 103 gene or a portion of thegene that directs the production of a normal 103 gene protein withnormal 103 gene function, can be inserted into cells, using vectorswhich include, but are not limited to adenovirus, adeno-associatedvirus, and retrovirus vectors, in addition to other particles thatintroduce DNA into cells, such as liposomes.

Such gene replacement techniques can be accomplished either in vivo orin vitro. As above, for 103 genes encoding extracellular molecules(e.g., a secreted 103 gene product), the cell type expressing the targetgene is less important than achieving a sufficient circulatingconcentration of the extracellular molecule for the amelioration ofimmune disorders, or to ameliorate one or more symptoms associated withan immune disorder described herein (e.g., asthma). Further, as above,when the gene encodes a gene product which acts intracellularly or as atransmembrane molecule, the gene must be expressed with the TH cellsubpopulation cell type of interest. Techniques which select forexpression within the cell type of interest are, therefore, preferredfor this latter class of target genes. In vivo, such techniques can, forexample, include appropriate local administration of target genesequences.

Additional methods which may be utilized to increase the overall levelof 103 gene expression and/or target and/or pathway gene activityinclude the introduction of appropriate 103 gene-expressing cells,preferably autologous cells, into a patient at positions and in numberswhich are sufficient to ameliorate the symptoms of T cell subpopulationrelated disorders. Such cells may be either recombinant ornon-recombinant. Among the cells which can be administered to increasethe overall level of 103 gene expression in a patient are normal cells,which express a 103 gene. The cells can be administered at theanatomical site of expression, or as part of a tissue graft located at adifferent site in the body. Such cell-based gene therapy techniques arewell known to those skilled in the art, see, e.g., Anderson et al., U.S.Pat. No. 5,399,349; Mulligan & Wilson, U.S. Pat. No. 5,460,959.

In vitro, target gene sequences can be introduced into autologous cells.These cells expressing the 103 gene sequence can then be reintroduced,preferably by intravenous administration, into the patient such thatthere results an amelioration of the symptoms of the disorder.

Alternatively, for the amelioration of a TH cell subpopulation-relateddisorder, TH cells belonging to a specific TH cell subpopulation (e.g.,a TH2 cell subpopulation) can be administered to a patient such that theoverall number of cells belonging to that TH cell subpopulation relativeto other TH cell subpopulation cells is increased. Techniques for suchTH cell subpopulation augmentation are described, below, in Section5.6.3.

5.5.3. NEGATIVE OR POSITIVE MODULATORY TECHNIQUES

Described hereinbelow are modulatory techniques which, depending on thespecific application for which they are utilized, can yield eitherpositive or negative responses leading to the amelioration of immunedisorders, including TH cell subpopulation-related disorders. Thus, inappropriate instances, the procedures of this Section can be used inconjunction with the negative modulatory techniques described, above, inSection 5.6.1, or, alternatively, in conjunction with the positiveregulatory techniques described, above, in Section 5.6.2.

Antibody Techniques:

Antibodies exhibiting modulatory capability can be utilized toameliorate immune disorders such as TH cell subpopulation-relateddisorders. Depending on the specific antibody, the modulatory effect canbe negative and can, therefore, be utilized as part of the techniquesdescribed, above, in Section 5.6.1, or can be positive, and can,therefore, be used in conjunction with the techniques described, above,in Section 5.6.2.

An antibody having negative modulatory capability refers to an antibodywhich specifically binds to and interferes with the action of a protein.For example, such an antibody could specifically bind the extracellulardomain of a transmembrane 103 gene product in a manner which does notactivate the 103 gene product but which disrupts the ability of the 103gene product to bind its natural ligand. Such antibodies can begenerated using standard techniques described in Section 5.3, above,against full length wild type or mutant 103 gene proteins, or againstpeptides corresponding to portions of the 103 gene proteins. Theantibodies include but are not limited to polyclonal, monoclonal, FAbfragments, single chain antibodies, chimeric antibodies, and the like.

An antibody having positive modulatory capability refers to an antibodywhich specifically binds to a protein and, by binding, serves to, eitherdirectly or indirectly, activate the function of the protein which itrecognizes. For example, an antibody can bind to the extracellularportion of a transmembrane protein, such as a 103 gene protein, in amanner which causes the transmembrane protein to function as though itsendogenous ligand was binding, thus activating, for example, a signaltransduction pathway. Such antibodies can also be generated usingstandard techniques described in Section 5.3, above, against full lengthwild type or mutant 103 gene proteins, or against peptides correspondingto portions of the 103 gene proteins. The antibodies include but are notlimited to polyclonal, monoclonal, human, humanized, FAb fragments,single chain antibodies, chimeric antibodies, and the like.

Preferably, the antibodies used in such modulatory techniquesspecifically recognize and/or bind to the extracellular domain of a 103gene product and need not be internalized in cells. However, in other,less preferred embodiments the antibodies may target or bind to, e.g.,intracellular domains of a 103 gene product and must, therefore, beinternalized. In such embodiments, lipofectin or liposomes can be usedto deliver the antibody or a fragment of the Fab region which binds tothe gene product epitope into cells. Where fragments of the antibody areused, the smallest inhibitory fragment which binds to the protein ispreferred. For example, peptides having an amino acid sequencecorresponding to the domain of the variable region of the antibody thatbinds to the protein can be used. Such peptides can be synthesizedchemically or produced via recombinant DNA technology using methods wellknown in the art (e.g., see Creighton, 1983, supra; and Sambrook et al.,1989, above). Alternatively, single chain antibodies, such asneutralizing antibodies, which bind to intracellular epitopes can alsobe administered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population by utilizing, for example,techniques such as those described in Marasco et al. (Marasco, W. etal., 1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

In more preferred embodiments, where the 103 gene protein to which theantibody is directed is extracellular (e.g., secreted), or is atransmembrane protein, any of the administration techniques described,below in Section 5.7 which are appropriate for peptide administrationcan be utilized to effectively administer the antibodies to their siteof action.

Increasing or Decreasing Specific TH Cell Subpopulation Concentrations:

Techniques described herein can be utilized to either deplete or augmentthe total number of cells belonging to a given TH cell subpopulation,thus effectively increasing or decreasing the ratio of the TH cellsubpopulation of interest to other TH cell subpopulations. Specifically,separation techniques are described which can be used to either depleteor augment the total number of cells present within a TH cellsubpopulation, and, further, targeting techniques are described whichcan be utilized to deplete specific TH cell subpopulations.

Depending on the particular application, changing the number of cellsbelonging to a TH cell subpopulation can yield either stimulatory orinhibitory responses leading to the amelioration of TH cellsubpopulation disorders. Thus, in appropriate instances, the proceduresof this Section can be used in conjunction with the inhibitorytechniques described, above, in Section 5.6.1 or, alternatively, inconjunction with the stimulatory techniques described, above, in Section5.6.2.

The separation techniques described herein are based on the presence orabsence of specific cell surface markers, preferably transmembranemarkers. Such markers can include, but are not limited to, theTH2-specific 103 gene product extracellular domain markers.

In instances wherein the goal of the separation is to increase oraugment the number of cells belonging to a specific TH cellsubpopulation, the surface markers used can also be surface markers thatare present on undifferentiated or partially undifferentiated TH cells.After separation, and purification of such undifferentiated or partiallydifferentiated TH cells, the cells can be cultured in physiologicalbuffer or culture medium and induced to differentiate by culturing inthe presence of appropriate factors. For example, IL-4 can be added toinduce the TH cells to differentiate into TH2 cells, while the cytokineIL-12 can be added to induce the TH cells to differentiate into TH1cells. After differentiation, cells can be washed, resuspended in, forexample, buffered saline, and reintroduced into a patient via,preferably, intravenous administration.

Separation techniques can be utilized which separate and purify cells,in vitro, from a population of cells, such as hematopoietic cellsautologous to the patient being treated. An initial TH cellsubpopulation-containing population of cells, such as hematopoieticcells, can be obtained using standard procedures well known to those ofskill in the art. Peripheral blood can be utilized as one potentialstarting source for such techniques, and can, for example, be obtainedvia venipuncture and collection into heparinized tubes.

Once the starting source of autologous cells is obtained, the T cells,such as TH1 or TH2 cells, can be removed, and thus selectively separatedand purified, by various methods which utilize antibodies which bindspecific markers present on the T cell population of interest, whileabsent on other cells within the starting source. These techniques caninclude, for example, flow cytometry using a fluorescence activated cellsorter (FACS) and specific fluorochromes, biotin-avidin orbiotin-streptavidin separations using biotin conjugated to cell surfacemarker-specific antibodies and avidin or streptavidin bound to a solidsupport such as affinity column matrix or plastic surfaces or magneticseparations using antibody-coated magnetic beads.

Separation via antibodies for specific markers, such as the TH2 specific103 gene product, can be by negative or positive selection procedures.In negative separation, antibodies are used which are specific formarkers present on undesired cells. For example, in the case of immunedisorders associated with a strong or inappropriate TH2 or TH2-likeimmune response, it can be desirable to deplete the number of TH2 cells.In such instances antibodies could be directed to the extracellulardomain of the 103 gene product. Cells bound by an antibody to such acell surface marker can be removed or lysed and the remaining desiredmixture retained.

In positive separation, antibodies specific for markers present on thedesired cells of interest. For example, in the case of certain immunedisorders that are associated with a weak or insufficient TH2 orTH2-like immune response it can be desirable to increase the number ofTH2 cells. In such instances antibodies could be directed to theextracellular domain of the 103 gene product. Cells bound by theantibody are separated and retained. It will be understood that positiveand negative separations can be used substantially simultaneously or ina sequential manner.

A common technique for antibody based separation is the use of flowcytometry such as by a florescence activated cell sorter (FACS).Typically, separation by flow cytometry is performed as follows. Thesuspended mixture of cells are centrifuged and resuspended in media.Antibodies which are conjugated to fluorochrome are added to allow thebinding of the antibodies to specific cell surface markers. The cellmixture is then washed by one or more centrifugation and resuspensionsteps. The mixture is run through a FACS which separates the cells basedon different fluorescence characteristics. FACS systems are available invarying levels of performance and ability, including multi-coloranalysis. The facilitating cell can be identified by a characteristicprofile of forward and side scatter which is influenced by size andgranularity, as well as by positive and/or negative expression ofcertain cell surface markers.

Other separation techniques besides flow cytometry can also provide fastseparations. One such method is biotin-avidin based separation byaffinity chromatography. Typically, such a technique is performed byincubating cells with biotin-coupled antibodies to specific markers,such as, for example, the transmembrane protein encoded by the 103 gene,followed by passage through an avidin column. Biotin-antibody-cellcomplexes bind to the column via the biotin-avidin interaction, whileother cells pass through the column. The specificity of thebiotin-avidin system is well suited for rapid positive separation.Multiple passages can ensure separation of a sufficient level of the THcell subpopulation of interest.

In instances whereby the goal of the separation technique is to depletethe overall number of cells belonging to a TH cell subpopulation, thecells derived from the starting source of cells which has now beeneffectively depleted of TH cell subpopulation cells can be reintroducedinto the patient. Such a depletion of the TH cell subpopulation resultsin the amelioration of TH cell subpopulation-related disordersassociated with the activity or overactivity of the TH cellsubpopulation. Reintroduction of the TH cell subpopulation-depletedcells can be accomplished by washing the cells, resuspending in, forexample, buffered saline, and intravenously administering the cells intothe patient.

If cell viability and recovery are sufficient, TH cellsubpopulation-depleted cells can be reintroduced into patientsimmediately subsequent to separation. Alternatively, TH cellsubpopulation-depleted cells can be cultured and expanded ex vivo priorto administration to a patient. Expansion can be accomplished via wellknown techniques utilizing physiological buffers or culture media in thepresence of appropriate expansion factors such as interleukins and otherwell known growth factors.

In instances whereby the goal of the separation technique is to augmentor increase the overall number of cells belonging to a TH cellsubpopulation, cells derived from the purified TH cell subpopulationcells can be reintroduced into the patient, thus resulting in theamelioration of TH cell subpopulation-related disorders associated withan under activity of the TH cell subpopulation.

The cells to be reintroduced will be cultured and expanded ex vivo priorto reintroduction. Purified TH cell subpopulation cells can be washed,suspended in, for example, buffered saline, and reintroduced into thepatient via intravenous administration.

Cells to be expanded can be cultured, using standard procedures, in thepresence of an appropriate expansion agent which induces proliferationof the purified TH cell subpopulation. Such an expansion agent can, forexample, be any appropriate cytokine, antigen, or antibody. In the caseof TH2 cells, for example, the expansion agent can be IL-4, while forTH1 cells, the expansion agent can, for example, be IL-12.

Prior to being reintroduced into a patient, the purified cells can bemodified by, for example, transformation with gene sequences encodinggene products of interest, including, but not limited to, gene sequencesencoding any of the 103 gene products described in Section 5.2 above.Such gene products should represent products which enhance the activityof the purified TH cell subpopulation or, alternatively, representproducts which repress the activity of one or more of the other TH cellsubpopulations. Cell transformation and gene expression procedures arewell known to those of skill in the art, and can be as those described,above, in Section 5.2.

Well known targeting methods can, additionally, be utilized in instanceswherein the goal is to deplete the number of cells belonging to aspecific TH cell subpopulation. Such targeting methods can be in vivo orin vitro, and can involve the introduction of targeting agents into apopulation of cells such that the targeting agents selectively destroy aspecific subset of the cells within the population. In vivoadministration techniques which can be followed for such targetingagents are described, below, in Section 5.7.

Targeting agents generally comprise, first, a targeting moiety which, inthe current instance, causes the targeting agent to selectivelyassociate with a specific TH cell subpopulation. The targeting agentsgenerally comprise, second, a moiety capable of destroying a cell withwhich the targeting agent has become associated.

Targeting moieties can include, but are not limited to, antibodiesdirected to cell surface markers found specifically on the TH cellsubpopulation being targeted, or, alternatively, to ligands, such asgrowth factors, which bind receptor-type molecules found exclusively onthe targeted TH cell subpopulation. In the case of TH2 cells, forexample, such a targeting moiety can represent an antibody directedagainst the extracellular portion of the 103 gene product describedherein, or can, alternatively, represent a ligand specific for thisreceptor-type TH2 specific molecule.

Destructive moieties include any moiety capable of inactivating ordestroying a cell to which the targeting agent has become bound. Forexample, a destructive moiety can include, but it is not limited tocytotoxins or radioactive agents. Cytotoxins include, for example,plant-, fungus-, or bacteria-derived toxins, with deglycosylated Ricin Achain toxins being generally preferred due to their potency and lengthyhalf-lives.

5.6. PHARMACEUTICAL PREPARATIONS AND METHODS OF ADMINISTRATION

The compounds, nucleic acid sequence and TH cell subpopulationsdescribed herein can be administered to a patient at therapeuticallyeffective doses to treat or ameliorate symptoms of immune disorders,including TH cell subpopulation related disorders; i.e., immunedisorders that are associated with an immune response of a particular THcell subpopulation. As used herein, a therapeutically effective doserefers to that amount of a compound (or of a TH cell subpopulation)sufficient to result in amelioration of a symptom or symptoms of theimmune disorder or, alternatively, to the amount of a nucleic acidsequence sufficient to express a concentration of a gene product whichresults in amelioration of symptoms of the immune disorders.

5.6.1. EFFECTIVE DOSE

Toxicity and therapeutic efficacy of compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a protein orpolypeptide (i.e., an effective dose or effective dosage) ranges fromabout 0.001 to 30 mg/kg of body weight, preferably about 0.01 to 25mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight,and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide or antibody can include asingle treatment or, preferably, can include a series of treatments. Ina preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weightone time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may also be apparent to one skilled in theart from the results of diagnostic assays as described herein.

5.6.2. FORMULATIONS AND METHODS OF USE

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvents can be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal,intralesional, vaginal or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration (i.e.,intravenous or intramuscular) by injection, via, for example, bolusinjection or continuous infusion. Formulations for injection can bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions can take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. It is preferredthat the TH cell subpopulation cells be introduced into patients viaintravenous administration.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Intralesional administration can comprise, for example, perfusing orcontacting a graft or organ with a composition prior to transplantation.

The compositions can, if desired, be presented in a pack or dispenserdevice which can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

5.6.3. PHARMACEUTICAL PREPARATIONS INCLUDING ANTIBODIES

Antibodies which specifically bind to 103 gene products of the inventionand thereby modulate their activity can also be administered to apatient at therapeutically effective doeses to treat or ameliorateimmune disorders. For example, Section 6.4, below, demonstrates the useof anti-103 gene product antibodies to reduce symptoms associated withasthma in an art-recognized animal model.

Antibodies of the invention are administered by any suitable means,including those described in Section 5.11.2, above. In addition,antibodies to a 103 gene product of the invention can be suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Preferably, the dosing is administered by injections, mostpreferably by intravenous or subcutaneous injections, depending in parton whether the administration is brief or chronic.

The appropriate dosage of antibody will depend on the type of disease tobe treated, the severity and course of the disease, whether the antibodyis administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the antibody,and the discretion of the attending physician. The antibody may suitablyadministered to the patient at one time or, more preferably, over aseries of treatments.

As a general proposition, the initial pharmaceutically effective amountof antibody administered parenterally will be in the range of about 0.1to 20 mg/kg of patient body weight per day, with the typical initialrange being 0.3 to 15 mg/kg of patient body weight per day. Where thesubsequent dosing is less than 100% of initial dosing, such subsequentdosing is calculated on the basis of daily dosing. Thus, for example, ifthe dosing regimen consists of daily injections of 2 mg/kg of patientbody weight per day for two weeks followed by a biweekly dose of 0.5mg/kg of patient body weight per day for 99 days, this would amount to asubsequent dose of about 1.8% of the initial dose calculated on a dailybasis (i.e., 2/day/100%=0.5/14 days/x %, x=1.8%). Preferably, thesubsequent dosing is less than about 50%, more preferably, less thanabout 25%, still more preferably, less than about 10%, and still morepreferably, less than about 5%. Most preferably, the subsequent dosingis less than about 2% of the initial dosing.

Overall, dosage ranges to be administered will, preferably, range fromabout 1 μg/kg to about 100 mg/kg, 1 μg/kg to about 15 mg/kg, or about 1μg/kg, to about 2.0 mg/kg body weight.

The preferred scheduling is that the initial dosing is administered noless frequently than daily, and up to an including continuously byinfusion. More preferably, depending on the specific disorder or injury,the initial daily dosing is administered for at least about one week,and preferably at least about two weeks. To obtain the most efficaciousresults, the initial dosing is preferably given as close to the firstsign, diagnosis, appearance, or occurrence of the disorder as possible,or, particular in the case of immune disorders, during remission of thedisorder.

Subsequent dosing is preferably administered periodically no more aboutonce a week. More preferably, the subsequent dosing is administered nomore than once biweekly. Subsequent dosing is typically administered forat least about five weeks, and preferably for at least about 10 weeks,after the initial dosing is terminated.

Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

Antibodies or antibodies conjugated to therapeutic moieties can beadministered to an individual alone or in combination with cytotoxicfactor(s), chemotherapeutic drug(s), and/or cytokine(s). If the latter,preferably, the antibodies are administered first and the cytotoxicfactor(s), chemotherapeutic drug(s) and/or cytokine(s) are administeredthereafter within 24 hours. The antibodies and cytotoxic factor(s),chemotherapeutic drug(s) and/or cytokine(s) can be administered bymultiple cycles depending upon the clinical response of the patient.Further, the antibodies and cytotoxic factor(s), chemotherapeuticdrug(s) and/or cytokine(s) can be administered by the same or separateroutes, for example, by intravenous, intranasal or intramuscularadministration. Cytotoxic factors include, but are not limited to,TNF-α, TNF-β, IL-1, IFN-γ and IL-2. Chemotherapeutic drugs include, butare not limited to, 5-fluorouracil (5FU), vinblastine, actinomycin D,etoposide, cisplatin, methotrexate and doxorubicin. Cytokines include,but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10 and IL-12.

Exemplary dosing regimens are disclosed in Jardieu, P. M., and Presta,L. G., 1998, WO 98/23761; and in Jardieu, P. M., and Presta, L. G.,1994, WO 94/04188, each of which is incorporated herein, by reference,in its entirety.

5.7. DIAGNOSTIC AND MONITORING TECHNIQUES

A variety of Methods can be employed for the diagnosis of immunedisorders, e.g., TH cell subpopulation-related disorders, predispositionto such immune disorders, for monitoring the efficacy of anti-immunedisorder compounds during, for example, clinical trials and formonitoring patients undergoing clinical evaluation for the treatment ofsuch disorders. Further, a number of methods can be utilized for thedetection of activated immune cells, e.g., activated members of a THcell subpopulation such as a TH2 or TH2-like cell subpopulation.

Such methods can, for example, utilize reagents such as the 103 genenucleotide sequences described in Sections 5.1, and antibodies directedagainst 103 gene peptides, as described, above, in Sections 5.2(peptides) and 5.3 (antibodies). Specifically, such reagents can beused, for example, for: (1) the detection of the presence of 103 geneexpression, 103 gene mutations, the detection of either over- orunder-expression of 103 gene mRNA relative to the non-immune disorderstate or relative to an unactivated TH cell subpopulation; (2) thedetection of aberrant expression (i.e., either an over- or anunder-abundance) of 103 gene product relative to the non-immune disorderstate or relative to the unactivated TH cell subpopulation state; and(3) the identification of specific TH cell subpopulation cells(including, for example, cells such as TH2 cells involved, e.g. in anatopic conditions such as asthma and allergy) within a mixed populationof cells.

The methods described herein can be performed, for example, by utilizingpre-packaged diagnostic kits comprising 103 gene nucleic acid oranti-103 gene antibody reagent described herein, which can beconveniently used, e.g., in clinical settings, to diagnose patientsexhibiting TH1- or TH2-related abnormalities.

Any cell type or tissue, preferably TH cells, in which the 103 gene isexpressed can be utilized in the diagnostics described below.

Among the methods which can be utilized herein are methods formonitoring the efficacy of compounds in clinical trials for thetreatment of immune disorders. Such compounds can, for example, becompounds such as those described, above, in Section 5.6. Such a methodcomprises detecting, in a patient sample, a gene transcript or geneproduct, such as a 103 gene transcript or a 103 gene product, which isdifferentially expressed in a TH cell subpopulation in an immunedisorder state relative to its expression in the TH cell subpopulationwhen the cell subpopulation is in a normal, or non-immune disorder,state.

Any of the nucleic acid detection techniques described hereinbelow, inSection 5.8.1 or any of the peptide detection techniques describedhereinbelow, in Section 5.8.2 can be used to detect a 103 genetranscript or gene product which is differentially expressed in theimmune disorder TH cell subpopulation relative to its expression in thenormal, or non-immune disorder, state.

During clinical trials, for example, the expression of the 103 gene canbe determined for the TH cell subpopulation in the presence or absenceof the compound being tested. The efficacy of the compound can befollowed by comparing the expression data obtained to the correspondingknown expression patterns for the TH cell subpopulation in a normal,non-immune disorder state. Compounds exhibiting efficacy are those whichalter the 103 gene expression of the immune disorder TH cellsubpopulation to more closely resemble that of the normal, non-immunedisorder TH cell subpopulation.

The detection of the product or products of genes, such as the 103 gene,that are differentially expressed in a TH cell subpopulation in animmune disorder state relative to their expression in the TH cellsubpopulation when the cell subpopulation is in a normal, or non-immunedisorder, state can also be used for monitoring the efficacy ofpotential anti-immune disorder compounds during clinical trials. Duringclinical trials, for example, the level and/or activity of the productsof a 103 gene can be determined for the TH cell subpopulation in thepresence or absence of the compound being tested. The efficacy of thecompound can be followed by comparing the 103 gene protein level and/oractivity data obtained to the corresponding known levels/activities forthe TH cell subpopulation in a normal, non-immune disorder state.Compounds exhibiting efficacy are those which alter the 103 gene proteinlevel and/or activity of the immune disorder TH cell subpopulation tomore closely resemble that of the normal, non-immune disorder TH cellsubpopulation.

Given the TH2-specific nature of the 103 gene, the detection of 103 genetranscripts and/or products can be particularly suitable for monitoringthe efficacy of compounds in clinical trials for the treatment of TH2cell subpopulation-related immune disorders such as, for example, asthmaor allergy.

Among the additional methods which can be utilized herein are methodsfor detecting TH cell responsiveness, for example, responsiveness toantigen, and for detecting activated immune cells, e.g., activatedmembers of TH cell subpopulations. Detection methods such as these areimportant in that many immune disorders involve inappropriate ratherthan insufficient immune responses. Such detection methods can be used,for example, to detect a predisposition to an immune disorder.

Methods for detecting TH cell responsiveness and/or activation cancomprise, for example, detecting in a TH cell sample a gene transcriptor product, such as 103 gene transcript or product, which isdifferentially expressed in TH cell subpopulation which is in anactivated or responsive state (e.g., a state in which the TH cellsubpopulation has been exposed to antigen), relative to a TH cellsubpopulation which is in an unactivated or nonresponsive state.

Any of the nucleic acid detection techniques described hereinbelow, inSection 5.8.1, or any of the peptide detection techniques describedhereinbelow, in Section 5.8.2 can be used to detect such adifferentially expressed gene transcript or gene product.

In addition to diagnostic uses, such techniques can also be utilized aspart of methods for identifying compounds which alter the cellularexpression of one or more of the differentially expressed genesdescribed herein, or as part of methods for identifying compounds whichalter the cellular and/or secreted level of product produced by thedifferentially expressed genes described herein. Such techniques can beused to identify compounds which alter the level of expression of the103 gene or the level of 103 gene product present in a cell. Suchmethods can include, for example, contacting a T cell with a compound,measuring the level of 103 gene expression in the cell (or the level of103 gene product in the cell), then comparing the level obtained to thatof a cell not exposed to the compound. The T cells used herein caninclude, for example, TH0, TH1 or TH2 cells. Such methods can furtherinclude stimulating the cells, for example, stimulating the cells priorto contacting the cells with the compound. Among the methods forstimulation are stimulation via anti-CD3 antibody stimulation.

Such methods can be performed such that the cell contacted is presentedwithin a non-human mammal, for example, a mouse. Further, among thenon-human mammals which can be utilized as part of these methods areones which exhibit symptoms of a T cell-related disorder (such as, forexample a TH2-related disorder, e.g., asthma), and contacting the cellwith the compound can ameliorate symptoms of the disorder.

The TH2-specific nature of the 103 gene can make the detection of itsgene transcripts and/or products particularly suitable for detectingactivation and/or responsiveness of TH2 cells.

5.7.1. DETECTING 103 GENE NUCLEIC ACIDS

DNA or RNA from the cell type or tissue to be analyzed can easily beisolated using procedures which are well known to those in the art.Diagnostic procedures can also be performed “in situ” directly upon, forexample tissue sections (fixed and/or frozen) of patient tissue obtainedfrom biopsies or resections, such that no nucleic acid purification isnecessary. Nucleic acid reagents such as those described in Section 5.1can be used as probes and/or primers for such in situ procedures (see,for example, Nuovo, G. J., 1992, “PCR In Situ Hybridization: Protocolsand Applications”, Raven Press, NY). Expression of specific cells withina population of cells can also be determined, via, for example, in situtechniques such as those described above, or by standard flow cytometrictechniques.

103 gene nucleotide sequences, either RNA or DNA, can, for example, beused in hybridization or amplification assays of biological samples todetect 103 gene structures and expression. Such assays can include, butare not limited to, Southern or Northern analyses, single strandedconformational polymorphism analyses, in situ hybridization assays, andpolymerase chain reaction analyses. Such analyses can reveal bothquantitative aspects of the expression pattern of the 103 gene, andqualitative aspects of the 103 gene expression and/or gene composition.That is, such techniques can detect not only the presence of 103 geneexpression, but can also detect the amount of expression, particularlywhich specific cells are expressing the 103 gene, and can, further, forexample, detect point mutations, insertions, deletions, chromosomalrearrangements, and/or activation or inactivation of 103 geneexpression.

Diagnostic methods for the detection of 103 gene-specific nucleic acidmolecules can involve for example, contacting and incubating nucleicacids, derived from the cell type or tissue being analyzed, with one ormore labeled nucleic acid reagents as are described in Section 5.1,under conditions favorable for the specific annealing of these reagentsto their complementary sequences within the nucleic acid molecule ofinterest. Preferably, the lengths of these nucleic acid reagents are atleast 15 to 30 nucleotides. After incubation, all non-annealed nucleicacids are removed from the nucleic acid:103 gene molecule hybrid. Thepresence of nucleic acids from the cell type or tissue which havehybridized, if any such molecules exist, is then detected. Using such adetection scheme, the nucleic acid from the tissue or cell type ofinterest can be immobilized, for example, to a solid support such as amembrane, or a plastic surface such as that on a microtiter plate orpolystyrene beads. In this case, after incubation, non-annealed, labelednucleic acid reagents of the type described in Section 5.1 are easilyremoved. Detection of the remaining, annealed, labeled 103 nucleic acidreagents is accomplished using standard techniques well-known to thosein the art.

Alternative diagnostic methods for the detection of 103 gene specificnucleic acid molecules can involve their amplification, e.g., by PCR(the experimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat.No. 4,683,202), ligase chain reaction (Barany, F., 1991, Proc. Natl.Acad. Sci. USA 88:189-193), self sustained sequence replication(Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),transcriptional amplification system (Kwoh, D. Y et al., 1989, Proc.Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. etal., 1988, Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment of such a detection scheme, a cDNA molecule isobtained from an RNA molecule of interest (e.g., by reversetranscription of the RNA molecule into cDNA). Cell types or tissues fromwhich such RNA can be isolated include any tissue in which 103 genesequences are known or suspected to be expressed, including, but notlimited, to TH0, TH1 and/or TH2 cell type-containing tissues. A sequencewithin the cDNA is then used as the template for a nucleic acidamplification reaction, such as a PCR amplification reaction, or thelike. The nucleic acid reagents used as synthesis initiation reagents(e.g., primers) in the reverse transcription and nucleic acidamplification steps of this method are chosen from among the 103 genenucleic acid reagents described in Section 5.1. The preferred lengths ofsuch nucleic acid reagents are at least 9-30 nucleotides. For detectionof the amplified product, the nucleic acid amplification can beperformed using radioactively or non-radioactively labeled nucleotides.Alternatively, enough amplified product can be made such that theproduct can be visualized by standard ethidium bromide staining or byutilizing any other suitable nucleic acid staining method.

TH1-related disorders can include, for example, chronic inflammatorydiseases and disorders, such as Crohn's disease, reactive arthritis,including Lyme disease, insulin-dependent diabetes, organ-specificautoimmunity, including multiple sclerosis, Hashimoto's thyroiditis andGrave's disease, contact dermatitis, psoriasis, graft rejection, graftversus host disease and sarcoidosis. TH2-related disorders can include,for example, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis) and certainviral infections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

Fingerprint patterns can be generated, for example, by utilizing adifferential display procedure, as discussed in U.S. patent applicationSer. No. 09/324,986 filed on Jun. 2, 1999 and in International PatentApplication No. WO 96/27603 published on Aug. 12, 1996 each of which isincorporated herein, by reference, in its entirety. Fingerprint patternscan also be generated, for example, by Northern analysis and/or RT-PCR.Any of the gene sequences described in either of the above-cited patentapplications and patent publication can be used as probes and/or RT-PCRprimers for the generation and corroboration of such fingerprintpatterns.

5.7.2. DETECTING 103 GENE PRODUCTS

Antibodies directed against wild type or mutant 103 gene peptides, whichare discussed, above, in Section 5.3, can also be used as TH cellsubpopulation-related disorder diagnostics and prognostics, asdescribed, for example, herein. Such diagnostic methods, can be used todetect 103 gene product, abnormalities in the level of 103 gene proteinexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of fingerprint gene protein.Structural differences can include, for example, differences in thesize, electronegativity, or antigenicity of the mutant fingerprint geneprotein relative to the normal fingerprint gene protein.

Protein from the tissue or cell type to be analyzed can easily beisolated using techniques which are well known to those of skill in theart. The protein isolation methods employed herein can, for example, besuch as those described in Harlow and Lane (Harlow, E. and Lane, D.,1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), which is incorporated herein byreference in its entirety.

In one embodiment, aberrant expression of a 103 polypeptide is detectedin a subject comprising: (a) contacting a sample of cells, tissue orbody fluid from said subject with an antibody or an antigen bindingfragment thereof; (b) measuring the level of the 103 polypeptide in saidsample, wherein an increase or decrease in the 103 polypeptide level insaid sample relative to a standard level of 103 polypeptide indicatesaberrant expression of said 103 polypeptide in said subject. This assaymay be performed, e.g., to monitor or detect an immune disorderdescribed herein. Preferably, the subject is an animal, more preferablya mammal and most preferably a human. It is also preferred that theantibody is a monoclonal antibody produced by the hyrbridoma clone M153F7.3, the hybridoma clone M15 2O3.1, the hybridoma clone M15 10F7.1,the hybridoma clone M15 1B4.1, the hybridoma clone M15 9F11.1, or thehybridoma clone M15 5A16.1. The term “standard level” refers to thelevel of expression of a 103 polypeptide by cells obtained from ahealthy subject or a subject without an immune disorder state, and/or tothe level of expression of a 103 polypeptide by a tissue sample or bodyfluid obtained from a healthy subject or a subject without an immunedisorder state. In particular, the term “standard level” can refer tothe level of expression of a 103 polypeptide by an unactivated TH cellsubpopulation state from a healthy subject. The level of expression of a103 polypeptide in a sample from a healthy subject or a subject withoutan immune disorder can be determined concomitantly with the test sampleor at an early time.

Preferred diagnostic methods for the detection of wild type or mutant103 gene peptide molecules can involve, for example, immunoassayswherein 103 gene peptides are detected by their interaction with ananti-fingerprint gene product-specific antibody.

For example, antibodies or fragments of antibodies, such as thosedescribed, above, in Section 5.3, useful in the present invention can beused to quantitatively or qualitatively detect the presence of wild typeor mutant 103 gene peptides. This can be accomplished, for example, byimmunofluorescence techniques employing a fluorescently labeled antibody(see below, this Section,) coupled with light microscopic, flowcytometric, or fluorimetric detection. Such techniques are especiallypreferred if the 103 gene peptides are expressed on the cell surface,such as, for example, is the case with the transmembrane form of 103gene product. Thus, the techniques described herein can be used todetect specific cells, within a population of cells, which express the103 gene product of interest.

The antibodies (or fragments thereof) useful in the present inventioncan, additionally, be employed histologically, as in immunofluorescenceor immunoelectron microscopy, for in situ detection of 103 genepeptides. In situ detection can be accomplished by removing ahistological specimen from a patient, and applying thereto a labeledantibody of the present invention. The antibody (or fragment) ispreferably applied by overlaying the labeled antibody (or fragment) ontoa biological sample. Through the use of such a procedure, it is possibleto determine not only the presence of the 103 gene peptides, but alsotheir distribution in the examined tissue. Using the present invention,those of ordinary skill will readily perceive that any of a wide varietyof histological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Immunoassays for wild type or mutant 103 gene peptides typicallycomprise incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells, or cells which have beenincubated in tissue culture, in the presence of a detectably labeledantibody capable of identifying 103 gene peptides, and detecting thebound antibody by any of a number of techniques well-known in the art.

The biological sample can be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support can then be washed with suitable buffersfollowed by treatment with the detectably labeled fingerprintgene-specific antibody. The solid phase support can then be washed withthe buffer a second time to remove unbound antibody. The amount of boundlabel on solid support can then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material can have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration can bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacecan be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of anti-wild type or mutant 103 geneproduct antibody can be determined according to well known methods.Those skilled in the art will be able to determine operative and optimalassay conditions for each determination by employing routineexperimentation.

One of the ways in which the 103 gene peptide-specific antibody can bedetectably labeled is by linking the same to an enzyme and use in anenzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked ImmunosorbentAssay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.); Voller, A. et al.,1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol.73:482-523; Maggio, E. (ed.), 1980, ENZYME IMMUNOASSAY, CRC Press, BocaRaton, Fla.; Ishikawa, E. et al., (eds.), 1981, ENZYME IMMUNOASSAY,Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Enzymes which can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect 103 gene wild type ormutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope (e.g., ¹²⁵I, ¹³⁵I, ³⁵S or ³H) can be detected by such means asthe use of a gamma counter or a scintillation counter or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wavelength, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

5.8 KITS

The invention encompasses kits for detecting the presence of apolypeptide or nucleic acid of the invention in a biological sample (atest sample). Such kits can be used to determine if a subject issuffering from or is at increased risk of developing a disorderassociated with aberrant expression of a polypeptide of the invention.For example, kits can be used to determine if a subject is sufferingfrom or is at increased risk of immune disorders such as Crohn'sdisease, reactive arthritis, Lyme disease, insulin-dependent diabetes,multiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, asthma and allergies (e.g., allergic rhinitis,gastrointestinal allergies such as food allergies), eosinophilia,conjunctivitis, glomerular nephritis, systemic lupus erythematosus,certain viral infections (e.g., HIV) and bacterial infections (e.g.,tuberculosis and lepromatous leprosy).

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to a 103polypeptide; and, optionally, (2) a second, different antibody whichbinds to either the polypeptide or the first antibody and is conjugatedto a detectable substance such as horseradish peroxidase, alkalinephosphatase, fluorescein, rhodamine, phycoerythrin, ¹²⁵I, ¹³¹I, ³²P, or³H (see, e.g., Section 5.3). The kit can also contain a control antibodywhich does not bind to the 103 polypeptide. Further, the kit can containa 103 polypeptide as a positive control for the antibodies of theinvention. Preferably, the kit comprises the monoclonal antibodyproduced by clone M15 3F7.3, clone M15 2O3.1, clone M15 10F7.1, cloneM15 1B4.1, clone M15 9F11.1, or clone M15 5A16.1. Each component of thekit is usually enclosed within an individual container and all of thevarious containers are within a single package. The kit may also includeinstructions for observing whether the tested subject is suffering fromor is at risk of developing a disorder associated with aberrantexpression of a 103 polypeptide.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a 103 polypeptide or (2)a pair of primers useful for amplifying a nucleic acid molecule encodinga 103 polypeptide. In a preferred embodiment, an oligonucleotide-basedkit comprises an oligonucleotide which hybridizes to the complement of anucleic acid molecule that encodes a polypeptide comprising the aminoacid sequence of SEQ ID NO:13. In another preferred embodiment, anoligonucleotide-based kit comprises a pair of primers which amplify anucleic acid molecule encoding the amino acid sequence of SEQ ID NO:13.The kit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions.

6. EXAMPLES

The following examples are presented as exemplary illustrations of themethods and compositions of the previously described invention and arenot limiting of that invention in any way. Specifically, the examplepresented in Section 6.1 describes the determination that the genereferred to herein as the 103 gene, which is also known as ST2, T1 andFit-1, is differentially expressed in TH cells. In particular, thisexample demonstrates that the 103 gene is expressed in TH2 and not inTH1 cells. The example in Section 6.2 describes the quantitativeexpression of human 103 in primary human cells. The example in Section6.3 describes the construction and expression of certain exemplary 103gene IgG1 fusion proteins, and the example in Section 6.4 describes theproduction of exemplary transgenic animals (transgenic mice) thatover-express the murine 103 gene product. The example presented inSection 6.5 demonstrates, using in vivo data from an art-recognizedanimal model, that the 103 gene product provides a critical signal toTH2 effector cells and can be utilized as a novel target for theselective suppression of TH2 immune response, e.g., in conditions suchas asthma and allergy. Thus, the in vivo data presented in Section 6.5demonstrates that molecules which target and/or inhibit 103 gene productactivity, such as anti-103 antibodies, are useful for treating immunedisorders, particularly TH cell subpopulation disorders such as asthmaand allergy. The example presented in Section 6.6 shows that the 103gene is also expressed in mammalian mast cells. The example presented inSection 6.7 describes additional experiments which confirms that the 103gene product is, not only a useful marker for identifying and detectingTH2 cells, but also play a crucial role in the differentiation andactivation of TH2, but not TH1, cells. The example presented in Section6.8 describes the production of human monoclonal antibodies against ahuman 103 gene product. Finally, the example presented in Section 6.9describes the detection of a ligand on mouse spleen cells whichspecifically binds to a 103 gene product.

Thus, the data presented hereinbelow show that the 103 gene product isan important regulator for a number of key events in both innate andadaptive immunity and that, as such, it is an important target for thetherapeutic and diagnostic methods and compositions of the presentinvention.

6.1. IDENTIFICATION AND CHARACTERIZATION OF A TH2-SPECIFIC GENE

In the Example presented in this Section, a transgenic T cell paradigmwas utilized to a identify a gene, referred to herein as the 103 gene,which is differentially expressed in TH2 cells. Specifically, this geneis present in TH2 cells while being completely absent from TH1 cells.The 103 gene, which corresponds to a gene known, alternatively, as ST-2T1 and Fit-1, does not appear to be expressed in any other assayed celltype or tissue, and is demonstrated herein to encode a marker which is,in vivo, completely TH2-specific. The 103 gene encodes a cell surfaceprotein, the potential significance of which is also discussed herein.

6.1.1. MATERIALS AND METHODS

Transgenic Mice:

Naive CD4⁺ cells were obtained from the spleens and/or lymph nodes ofunprimed transgenic mouse strains harboring a T cell receptor (TCR)recognizing ovalbumin (Murphy et al., 1990, Science 250:1720).

Ova-Specific Transgenic T Cells:

Suspensions of ova-specific T cells were co-cultured with stimulatorypeptide antigen and antigen presenting cells essentially as described inMurphy et al. (Murphy et al., 1990, Science 250:1720). Briefly, 2-4×10⁶T cells were incubated with approximately twice as many TA3 antigenpresenting cells in the presence of 0.3 μM Ova peptide. TH1 culturescontained approximately 10 ng/ml recombinant mIL-12. Conversely, TH2cells received IL-4 (1000 u/ml). Cultures were harvested at various timepoints after initiation of culture. T cells were purified of TA3 cellsusing anti-CD4 coated magnetic beads (Dynal, Inc.). T cells werepelleted by gentle centrifugation and lysed in the appropriate volume ofRNAzol™ (Tel-Test, Friendswood, Tex.).

Tissue Collection and RNA Isolation:

Cells were quick frozen on dry ice. Samples were then homogenizedtogether with a mortar and pestle under liquid nitrogen.

Total cellular RNA was extracted from tissue with either RNAzol™ orRNAzolB™ (Tel-Test, Friendswood, Tex.), according to the manufacturer'sinstructions. Briefly, the tissue was solubilized in an appropriateamount of RNAzol™ or RNAzolB™, and RNA was extracted by the addition of1/10 v/v chloroform to the solubilized sample followed by vigorousshaking for approximately 15 seconds. The mixture was then centrifugedfor 15 minutes at 12,000 g and the aqueous phase was removed to a freshtube. RNA was precipitated with isopropanol. The resultant RNA pelletwas dissolved in water and re-extracted with an equal volume ofchloroform to remove any remaining phenol. The extracted volume wasprecipitated with 2 volumes of ethanol in the presence of 150 mM sodiumacetate. The precipitated RNA was dissolved in water and theconcentration determined spectroscopically (A₂₆₀).

Differential Display:

Total cellular RNA (10-50 was treated with 20 Units DNase I (BoehringerMannheim, Germany) in the presence of 40 Units ribonuclease inhibitor(Boehringer Mannheim, Germany). After extraction with phenol/chloroformand ethanol precipitation, the RNA was dissolved in DEPC (diethylpyrocarbonate)-treated water. RNA (0.4-2 μg) was reverse-transcribedusing Superscript reverse transcriptase (GIBCO/BRL). The cDNAs were thenamplified by PCR on a Perkin-Elmer 9600 thermal cycler. The reactionmixtures (20 μL) included arbitrary decanucleotides and one of twelvepossible T₁₁ VN sequences, wherein V represents either dG, dC, or dA,and N represents either dG, dT, dA, or dC. Parameters for the 40 cyclePCR were as follows: Hold 94° C. 2 minutes; Cycle 94° C. 15 seconds, 40°C. 2 minutes; Ramp to 72° C. 30 seconds; Hold 72° C. 5 minutes; Hold 4°C.

Radiolabelled PCR amplification products were analyzed byelectrophoresis on 6% denaturing polyacrylamide gels.

Reamplification and Subcloning:

PCR bands of interest were recovered from sequencing gels andreamplified.

Briefly, autoradiograms were aligned with the dried gel, and the regioncontaining the bands of interest was excised with a scalpel. The excisedgel fragment was eluted by soaking in 100 μL TE (Tris-EDTA) buffer atapproximately 100° C. for 15 minutes. The gel slice was then pelleted bybrief centrifugation and the supernatant was transferred to a newmicrocentrifuge tube. DNA was combined with ethanol in the presence of100 mM Sodium acetate and 30 μg glycogen (Boerhinger Mannheim, Germany)and precipitated on dry ice for approximately 10 minutes. Samples werecentrifuged for 10 minutes and pellets were washed with 80% ethanol.Pellets were resuspended in 10 μL distilled water.

5 μL of the eluted DNA were reamplified in a 100 μL reaction containing:standard Cetus Taq polymerase buffer, 20 μM dNTPs, 1 μM of each of theoligonucleotide primers used in the initial generation of the amplifiedDNA. Cycling conditions used were the same as the initial conditionsused to generate the amplified band, as described above. One-half of theamplification reaction was run on a 2% agarose gel and eluted usingDE-81 paper (Whatman Paper, Ltd., England) as described in Sambrook etal., supra. Recovered fragments were ligated into the cloning vectorpCR™ II (Invitrogen, Inc., San Diego Calif.) and transformed intocompetent E. coli strain DH5α (Gibco/BRL, Gaithersburg, Md.). Colonieswere grown on LB-agar plates containing ampicillin (100 μg/mL) and X-gal(40 μg/mL) to permit blue/white selection.

Sequence Analysis:

After subcloning, reamplified cDNA fragments were sequenced on anApplied Biosystems Automated Sequencer (Applied Biosystems, Inc.Seattle, Wash.). Sequence was obtained from four or more independenttransformants containing the same insert. The nucleotide sequence shownherein represents either the consensus of the information obtained fromthe four sequences, or the sequence obtained from a representativeclone, as indicated. Such primary sequence data was edited and trimmedof vector sequences and highly repetitive sequences and used to searchGenbank databases using the BLAST (Altschul, S. F. et al., 1990, J. Mol.Biol. 215:403-410) program.

Northern Analysis:

RNA samples were electrophoresed in a denaturing agarose gel containing1-1.5% agarose (SeaKem™ LE, FMC BioProducts, Rockland, Me.) containing6.3% formaldehyde. Samples containing 5-20 μg of total RNA were mixedwith denaturing loading solution (72% deionized formamide andbromophenol blue) and heated to 70° C. for 5 minutes. Samples wereplaced on ice and immediately loaded onto gels. Gels were run in 1×MOPSbuffer (100 mM MOPS, 25 mM sodium acetate, 5 mM EDTA). Afterelectrophoresis, the gels were stained with ethidium bromide andvisualized with ultraviolet light.

After completion of electrophoresis, gels were soaked in 50 mM sodiumhydroxide with gentle agitation for approximately 30 minutes to lightlycleave RNA. Gels were rinsed twice in water and then neutralized bysoaking in 0.1 M Tris-HCl (pH 7.5) for approximately 30 minutes. Gelswere briefly equilibrated with 20×SSC (3M sodium chloride, 0.3 M sodiumcitrate) and then transferred to nylon membranes such as Hybond™, -N,(Amersham, Inc., Arlington Heights, Ill.) or Zeta-Probe (Bio-Rad, Inc.,Hercules, Calif.) overnight in 20×SSC. Membranes containing transferredRNA were baked at 80° C. for 2 hours to immobilize the RNA.

DNA fragments to be used as probes were of various sizes and werelabeled using a random hexamer labeling technique. Briefly, 25 ng of apurified DNA fragment was used to generate each probe. Fragments wereadded to a 20 μL random hexanucleotide labeling reaction (BoehringerMannheim, Inc., Indianapolis, Ind.) containing random hexamers and a mixof the nucleotides dCTP, dGTP, and dTTP (at a final concentration of 25μM each). The reaction mix was heat-denatured at 100° C. for 10 minutesand then chilled on ice. 5 μL of α-³²P-dATP (50 μCi; Amersham, Inc.,Arlington Heights, Ill.) and Klenow DNA polymerase (2 units; BoehringerMannheim, Inc., Indianapolis, Ind.) were added. Reactions were incubatedat 37° C. for 30 minutes. Following incubation, 30 μL water was added tothe labeling reaction and unincorporated nucleotides were removed bypassing the reactions through a BioSpin-6™ chromatography column(Bio-Rad, Inc., Hercules, Calif.). Specific incorporation was determinedusing a scintillation counter. 1-5×10⁶ cpm were used per mlhybridization mixture.

Nylon membranes containing immobilized RNA were prehybridized accordingto manufacturer's instructions. Radiolabelled probes were heat denaturedat 70° C. in 50% deionized formamide for 10 minutes and ten added to thehybridization mixture (containing 50% formamide, 10% dextran sulfate,0.1% SDS, 100 μg/mL sheared salmon sperm DNA, 5×SSC, 5×Denhardt'ssolution, 30 mM Tris-HCl (pH 8.5), 50 mM NaPO₄ (pH 6.5). Hybridizationswere carried out at 42° C. overnight. Nylon membranes were then bathedfor 2 minutes in a wash solution of 0.2×SSC and 0.1% SDS at roomtemperature to remove most of the remaining hybridization solution. Themembranes were then bathed twice in fresh 42° C. preheated wash solutionfor 20 minutes. Filters were covered in plastic wrap and exposed toautoradiographic film to visualize results.

RT-PCR Analysis:

Quantitative RT-PCR was performed as follows. 1-2 μg of total RNA,prepared as described above, was reverse transcribed with oligodT₍₁₂₋₁₈₎ primers and Superscript™ RNAase H⁻ reverse transcriptase(Gibco-BRL, Gaithersburg, Md.). Briefly, RNA was combined with 1 μLoligo dT (500 μg/mL) in a total volume of 11 μL. The mixture was heatedto 70° C. for 10 minutes and chilled on ice. After a briefcentrifugation, RNA was reverse transcribed for 1 hour. Aliquots of thefirst strand cDNA were stored at −20° C. until just prior to use.

Expression levels were determined by PCR amplification of serialdilutions of first strand cDNA. In this procedure, cDNA is seriallydiluted in water. The dilutions are then batch amplified by PCR usingsequence-specific primers. All PCR reactions are amplified underidentical conditions. Therefore, the amount of product generated shouldreflect the amount of sequence template which was initially present.5-10 fold dilutions of cDNA were used and enough dilutions were usedsuch that the amount of product subsequently produced ranged fromclearly visible, by UV illumination of ethidium bromide-stained gels, tobelow detection levels. The method described herein can distinguish10-fold differences in expression levels.

Primers were designed for the amplification of the sequenced amplifiedbands, which were chosen using the program OLIGO (National Biosciences,Plymouth, Minn.). Primer sequences used in this assay were as follows:

103 sense primer: (SEQ ID NO: 14) 5′-TTGCCATAGAGAGACCTC-3′;band 103 antisense primer: (SEQ ID NO: 15) 5′-TGCTGTCCAATTATACAGG-3′;murine gamma actin sense primer: (SEQ ID NO: 16)5′-GAACACGGCATTGTCACTAACT-3′; murine gamma actin antisense primer:(SEQ ID NO: 17) 5′-CCTCATAGATGGGCACTGTGT-3′.

All quantitative PCR reactions were carried out in a 9600 Perkin-ElmerPCR machine (Perkin-Elmer). Generally, amplification conditions were asfollows: 30-40 cycles consisting of a 95° C. denaturation for 30seconds, 50-60° C. annealing for 30 seconds, and 72° C. extension for 1minute. Following cycling, reactions were extended for 10 minutes at 72°C.

RNase Protection Assays:

RNAse protection assays were performed according to manufacturer'sinstructions, using a kit purchased from Ambion, Inc. RNA probes derivedfrom GenBank Accession No. Y07519 were utilized in the RNAse protectionassays. These probes were also generated according to manufacturer'sinstructions, using a kit purchased from Ambion, Inc. The sequence ofthese RNA probes corresponds to the 5′ end of the gene, and includesboth coding and 5′ untranslated sequences.

Anti CD-3 Stimulation:

T cell clone pardigm searches were conducted using three differentclones: D10.G4 (TH2), AE7 (TH1) and D1.1 (TH1). Prior to stimulation,cell cultures were enriched for live cells by centrifugation through aFicoll gradient. Recovered cells were counted and their viability wasexamined using trypan blue exclusion. Cells were replated into eitherT25 or T75 flasks at approximately 5×10⁶ cells in 5 mLs or 1.5×10⁶ cellsin 10 mLs of culture medium, respectively.

Coating was performed, generally, according to Current ProtocolsImmunology, 1992, Coligan, J. E. et al., John Wiley & Sons, NY, pp3.12.4-3.12.6. Specifically, prior to plating, the flasks were coatedwith anti-CD3-ε antibodies (hybridoma supernatant from the 145-C11hybridoma; Parmingen, Inc., San Diego Calif.). For coating, antibodieswere resuspended in PBS at 1-2 μg/mL at a volume sufficient to coat thebottom of the flasks. Coating solution was incubated on the flasks forat least one hour at 37° C.

After incubation, the antibody coating solution was removed byaspiration and cells were immediately added. Flasks were placed in a 37°C. incubator for 6 hours. Cells were harvested by, for example, removalof supernatant from the culture followed by direct lysing of cells byaddition of RNAzol™ solution.

6.1.2. RESULTS

A differential display analysis of RNA isolated from TH1 and TH2 cellsamples obtained from a transgenic T cell paradigm study. Specifically,TH cells were obtained from transgenic mice harboring a T cell receptorrecognizing ovalbumin (Murphy et al., 1990, Science 250:1720) werestimulated three times, and RNA was obtained from TH1 and TH2 cells.Differential display analysis of the RNA samples resulted in theidentification of a TH2 differentially expressed band, designated andreferred to herein as band 103. The gene corresponding to band 103 isreferred to herein as the 103 gene.

103 gene cDNA was isolated, amplified and subcloned, and nucleotidesequence (SEQ ID NO:1) was obtained, as shown in FIG. 1. A databasesearch revealed that the nucleotide sequence of band 103 resulted in analignment with 98% identity to the mouse form of a gene known,alternatively, as the ST-2, T1 or Fit-1 gene (Klemenz, R. et al., 1989,Proc. Natl. Acad. Sci. USA 86:5708-5712; Tominaga, S., 1989, FEBS Lett.258:301-301; Werenskiold, A. K. et al., 1989, Mol. Cell. Biol.9:5207-5214; Werenskiold, A. K., 1992, Eur. J. Biochem. 204:1041-1047;Yanagisawa, K. et al., 1993, FEBS Lett. 318:83-87; Bergers, G. et al.,1994, EMBO J. 13:1176-1188).

The 103 gene encodes, possibly via alternatively spliced transcripts,transmembrane and soluble forms of proteins which belong to theimmunoglobulin superfamily. The soluble form of the protein shows a highlevel of similarity to the extracellular portion of the mouseinterleukin-1 receptor type 1 (IL-1R1) and interleukin-1 receptor type 2(IL-1R2; which lacks a cytoplasmic domain), while the transmembraneportion (termed ST2L) bears a high resemblance to the entire IL-1R1sequence and to the extracellular IL-1R2 sequences. Further, the 103gene appears to be tightly linked to the interleukin 1 receptor-type 1locus (McMahan, C. J. et al., 1991, EMBO J. 10:2821-2832; Tominaga, S.et al., 1991, Biochem. Biophys. Acta. 1090:1-8). Additionally, the human103 gene homolog has also been reported (Tominaga, S. et al., 1992,Biochem. Biophys. Acta. 1171:215-218). FIG. 2 illustrates the 103 genetransmembrane and soluble forms of protein, and shows their relationshipto the IL-1R1 protein sequence.

A quantitative RT-PCR analysis (FIG. 12) of RNA obtained from cells of aTH1 and TH2 cells, generated as described above, 24 hours after tertiaryantigen stimulation not only confirmed the putative TH2 differentialexpression of the gene, but, revealed that the expression of the 103gene appears to be TH2 specific, i.e., the sensitive RT-PCR studydetected no 103 gene message in the TH1 RNA sample.

The TH2 specificity of the 103 gene was further confirmed by a Northernanalysis of several representative TH cell lines. Specifically, threeTH2 clones (CDC25, D10.G4, DAX) and three TH1 clones (AE7.A, Dorris,D1.1) were utilized and RNA samples were isolated from eitherunstimulated cells or from cells which had been stimulated for 6 hourswith plate-bound anti-CD3 antibody. The samples were probed with band103 sequences, as shown in FIG. 13. While 103 gene RNA is present in RNAobtained from both unstimulated and stimulated cells of each of the TH2cell lines, 103 gene RNA is completely absent from all of the samplesobtained from either stimulated or unstimulated TH1 cells. As the RT-PCRanalysis described above first demonstrated, the 103 gene appears to beTH2 specific, with no detectable TH1-derived signal being present.

The data presented in FIG. 14 represent an additional Northern analysisin which 103 gene expression was assayed in TH cell clones (lanes 1-5)and in murine tissues (lanes 6-10). In addition to corroborating theexpression of 103 gene RNA in both stimulated and unstimulated TH2cells, the data presented here demonstrate that 103 gene expressionappears to be negative in each of the tissues (i.e., brain, heart, lung,spleen, and liver) tested.

FIG. 15 illustrates an RNAse protection assay which demonstrates twopoints regarding 103 gene regulation. First, this analysis of TH cellclones confirms the TH2-specific results described, above. Specifically,the results of this study demonstrate by RNase protection, that 103 genemRNA is absent from the TH1 clone AE7, but is present in the TH2 cloneD10.G4.

Second, RNAse protection revealed that alternate forms of 103 genetranscripts are produced upon stimulation of TH2 clones. Specifically,within 6 hours of anti-CD3 stimulation, two additional forms of 103 genetranscript appear in TH2 clones. These additional 103 gene transcriptforms represent, one, a transcript encoding a shortened, secreted,soluble form of the band 103 gene product, and, two, a smaller, termedmini, transcript which encodes a yet shorter form of the gene product.Thus, it appears that, while the 103 gene transcript encoding thetransmembrane gene product is expressed in both unstimulated andstimulated TH2 cells, the two shorter forms of transcript are expressedin a TH2-specific inducible manner. Further, while the 103 genetranscript encoding the transmembrane product are expressed in bothstimulated and unstimulated TH2 cells, the level of this transcriptpresent in stimulated is lower, i.e., is downregulated. Thus, the lowerlevel of transmembrane product and higher level of secreted 103 geneproduct can act synergistically to dampen some stimulation-inducedsignal transduction event.

Additionally, it should be noted that the results presented hereinrepresent the first time the mini form of 103 gene transcript, which canencode a shorter version of the soluble form of 103 gene product, hasbeen observed.

To summarize, while 103 gene expression in T helper cell lines hadpreviously been reported (Tominaga, S. et al., 1992, Biochem. Biophys.Acta. 1171:215-218), the TH paradigm/differential display techniquesutilized here have demonstrated, for the first time, that the 103 geneencodes a TH2 cell subpopulation-specific surface marker. In fact, theresults described in this Example demonstrate that the firstidentification of any in vivo TH cell subpopulation-specific cellularmarker.

Given its status as both a TH2 cell subpopulation-specific marker andcell surface protein, the full length 103 gene product can be utilizedin a variety of methods to modulate TH cell subpopulation-relateddisorders and/or to identify compounds which exhibit such modulatorycapability. The truncated forms of the 103 gene products can,additionally, be used as part of these methods. Modulatory methods aredescribed, above, in Section 5.6, while strategies for theidentification of modulatory compounds are described, above, in Section5.4.

6.2. EXPRESSION OF HUMAN 103 GENE PRODUCT

In the Example presented in this Section, primary human cells wereanalyzed for their quantitative expression of human 103.

6.2.1 MATERIALS AND METHODS

Primary Cells Analyzed:

cDNA was prepared from the following primary cells: resting andphytohemaglutinin (PHA) activated peripheral blood mononuclear cells(PBMC); resting and PHA activated CD3⁺ cells; CD4⁺ and CD8⁺ T cells;resting Th0, Th1 and Th2 cells; Th0, Th1 and Th2 cells stimulated for 1,6, 24 or 48 hours with anti-CD3 antibody; resting and lipopolysaccharide(LPS) activated CD19⁺ B cells; CD14⁺ cells; granulocytes; eosinophils;PBMC stimulated with IL-10 and IL-4; and PBMC stimulated withinterferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). cDNA was alsoprepared from two lung biopsies (referred to as biopsy #1 and biopsy #2)obtained from two asthmatic individuals 24 hours after exposure to anantigen (i.e., house dust mites).

RNA Isolation:

Cells were quick frozen on dry ice. Samples were then homogenizedtogether with a mortar and pestle under liquid nitrogen.

Total cellular RNA was extracted from tissue with either RNAzol™ orRNAzolB™ (Tel-Test, Friendswood, Tex.), according to the manufacturer'sinstructions. Briefly, the tissue was solubilized in an appropriateamount of RNAzol™ or RNAzolB™, and RNA was extracted by the addition of1/10 v/v chloroform to the solubilized sample followed by vigorousshaking for approximately 15 seconds. The mixture was then centrifugedfor 15 minutes at 12,000 g and the aqueous phase was removed to a freshtube. RNA was precipitated with isopropanol. The resultant RNA pelletwas dissolved in water and re-extracted with an equal volume ofchloroform to remove any remaining phenol. The extracted volume wasprecipitated with 2 volumes of ethanol in the presence of 150 mM sodiumacetate. The precipitated RNA was dissolved in water and theconcentration determined spectroscopically (A₂₆₀).

RT-PCR Analysis:

Probes were designed by PrimerExpress software (PE Biosystems) based onthe human 103 sequence. The primers and probes for expression analysisof human 103 and β-2 microglobulin were as follows:

103 Forward Primer (SEQ ID NO: 18) TGTGACGGCGACCAGGT 103 Reverse Primer(SEQ ID NO: 19) TCTCTGTTTCCAGTAATCGGAGC 103 TaqMan Probe (SEQ ID NO: 20)TTCACGGTCAAGGATGAGCAAGCCTT β-2 microglobulin Forward Primer(SEQ ID NO: 21) CACCCCCACTGAAAAAGATGA β-2 microglobulin Reverse Primer(SEQ ID NO: 22) CTTAACTATCTTGGGCTGTGACAAAG β-2 microglobulin TaqMan Probe (SEQ ID NO: 23) TATGCCTGCCGTGTGAACCACGTG 

The human 103 sequence probe was labeled using FAM(6-carboxyfluorescein), and the β2-microglobulin reference probe waslabeled with a different fluorescent dye, VIC. The differential labelingof the target human 103 sequence and internal reference gene thusenabled measurement in the same well. Forward and reverse primers andthe probes for both β2-microglobulin and the target human 103 sequencewere added to the TaqMan® Universal PCR Master Mix (PE AppliedBiosystems). Although the final concentration of primer and probe couldvary, each was internally consistent within a given experiment. Atypical experiment contained 200 nM of forward and reverse primers plus100 nM probe for β-2 microglobulin and 600 nM forward and reverseprimers plus 200 nM probe for the target h16395 sequence. TaqMan matrixexperiments were carried out on an ABI PRISM 7700 Sequence DetectionSystem (PE Applied Biosystems). The thermal cycler conditions were asfollows: hold for 2 min at 50° C. and 10 min at 95° C., followed bytwo-step PCR for 40 cycles of 95° C. for 15 sec followed by 60° C. for 1min.

The following method was used to quantitatively calculate human 103expression in the various cells relative to β-2 microglobulin expressionin the same cells. The threshold cycle (Ct) value is defined as thecycle at which a statistically significant increase in fluorescence isdetected. A lower Ct value is indicative of a higher mRNA concentration.The Ct value of the h16395 sequence is normalized by subtracting the Ctvalue of the β-2 microglobulin gene to obtain a Ct value using thefollowing formula: Ct=Ct_(h16395)−Ct_(β-2 microglobulin). Expression isthen calibrated against a cDNA sample showing a comparatively low levelof expression of the h16395 sequence. The Ct value for the calibratorsample is then subtracted from Ct for each tissue sample according tothe following formula: Ct=Ct-_(sample)−Ct-_(calibrator). Relativeexpression is then calculated using the arithmetic formula given by2^(−Ct).Expression of the target human 103 sequence in each of thetissues tested was then graphically represented as discussed in moredetail below.

6.2.2. RESULTS

FIG. 16 shows expression of the soluble and transmembrane forms of human103 as determined in a broad panel of cells as described above, relativeto human 103 expression in resting CD14+ cells. The results indicatethat expression of human 103 was detectable in resting Th0 cells, Th0stimulated for 1 and 6 hours with anti-CD3 antibody, Th1 stimulated for1, 6, and 48 hours with anti-CD3 antibody, TH2 cells, TH2 cellsstimulated for 1, 6 and 48 hours with anti-CD3 antibody, eosinophils,granulocytes, resting PBMC, and PBMC stimulated with IL-10 and IL-4. Thehigh expression observed among the hematopoietic cell populationsanalyzed was seen in resting and stimulated Th2 cells. The pattern ofexpression indicates that mRNA synthesis of human 103 transientlyincreases following stimulation with anti-CD3 antibody. The results alsoindicate that expression of human 103 was detectable in biopsiesobtained from the asthmatic individuals exposed to antigen.

6.3. CONSTRUCTION AND EXPRESSION OF 103 GENE IgG1 FUSION PROTEINS

Described in this example is the construction and expression of IgG1proteins. Specifically, the construction of exemplary murine 103 geneIgG1 fusion proteins is described.

6.3.1. MATERIALS AND METHODS

Generation of the Vector Encoding the Marine 103 Gene-hIg G1 FusionProtein:

The construction of a vector encoding a soluble Ig-fusion protein (size:approximately 60 kD) containing a murine 103 gene product extracellulardomain (but lacking the 103 gene product signal sequence) wasconstructed as described here. The CD44 portion of the pCD5-CD44-IgG1vector (described above) was replaced with a nucleotide sequenceencoding the 103 gene product extracellular domain. The 103 gene productextracellular domain sequence of the Ig-fusion protein consisted of 103gene product amino acid residues 27-342 (i.e., the 103 gene productportion ending with amino acid sequence Ile-Val-Ala-Gly-Cys-Ser).

The fragment encoding the 103 gene product extracellular domain wasamplified by PCR using synthetic oligonucleotides complementary to thesequences flanking the 103 gene region that would produce the 103 geneproduct containing amino acid residues 27-341. The oligonucleotides weredesigned to allow creation of a KpnI site at the 5′ end and a BamHI siteat the 3′ end of each amplified 103 gene fragment to facilitatesubsequent insertion into pCD5-CD44-IgG1.

The 5′ oligonucleotide was as follows:

(SEQ ID NO: 24) 5′-CCGCGGGTACCAGTAAATCGTCCTGGGGTGG-3′.

The 3′ oligonucleotide was as follows

(SEQ ID NO: 25) 5′-AAATAAAGGATCCCTACATCCAGCAACTATGTAGTA-3′.

PCR reaction conditions consisted of 15 cycles of 30 seconds at 95° C.,30 seconds at 60° C., and 30 seconds at 72° C., using Vent DNApolymerase (New England Biolabs, Beverly, Mass.) and 103L gene astemplate.

103 PCR products were digested with KpnI and BamHI, and ligated toKpnI-BamHI sites of CD5-IgG1 vector, thus replacing the CD44 sequenceswith the 103 gene sequences.

The resulting plasmid, encoding a fusion protein containing CD5-signalsequence, murine 103-extracellular domain and human-IgG1 heavy chain Fcregion, was transfected into COS cells using LipofectAMINE™ (GIBCOBRL,MD) following manufacturer's suggestions. 0.18 μg plasmid DNA and 140 μlLipofectAMINE™ were used for transfection of the cells of a 150 mmplate. Twenty-four hours after transfection, medium was replaced with10% Ultra-low IgG Fetal Bovine Serum (GIBCOBRL, MD)/DMEM (BioWHITTAKER,Maryland), and the transfected cells were allowed to grow for 4-5 dayscontinuously. Supernatants were then harvested, centrifuged to removenonadherent cells and debris, and stored at −20° C.

For purification, 1 ml of supernatant was precipitated overnight with 1μl of IPA-300 Immubilized rProteinA (Repligen, Mass.) at 4° C. The nextday, beads were collected by centrifugation and washed three times with10 volumes of PBS. For analysis, the beads were suspended in 20 μl of 2×Laemmli Sample Buffer (BIO-RAD, CA) and boiled at 100° C. for 10 min.The boiled sample was spun briefly and loaded onto a 10% SDS-PAGE gel(JILEinc. CT).

Metabolic Labelling of Recombinant Fusion Protein:

36 hours after transient transfection of COS-7 cultures, cells wererinsed with replacement growth medium [DMEM methionine and cysteinedepleted (ICN, Inc., CA)]. After rinsing, 150 μCI/ml medium of a mixtureof ³⁵S-cysteine and ³⁵S-methionine (Express ³⁵S³⁵S™, Dupont, Mass.) wasadded to the replacement medium and the cells were cultured overnight.

Analysis of Recombinant Protein by SDS Page:

hIgG1 fusion proteins were generated by LipfectAMINE™ (Gibco, Inc.,MD)-mediated transient transfection of COS-7 cells according tomanufacturer's suggestion for 200 gene-hIgG1 fusion proteins, 1 ml ofday 5 supernatant was mixed with 20 μl of Protein A Trisacryl bead(Pierce, Inc., IL) in the presence of 20 mM HEPES (pH 7.0) overnight at4° C. with constant agitation. Beads were then washed 3× with PBS priorto the addition to loading buffer. Beads were mixed with either reducingor non-reducing loading buffers (described in, Molecular Cloning,Sambrook, Fritsch, and Maniatis, 2nd edition, 1989, with the exceptionthat DTT was replaced with 2.5% β-mercaptoethanol).

6.3.2. RESULTS

The construction and expression of recombinant IgG fusion proteins isdescribed herein. Specifically, an exemplary 103 gene product-IgG1fusion protein is described. The 103 gene product-IgG1 fusion proteincontains a CD5 signal sequence and 103 gene product extracellular domainfused to a human IgG1 heavy chain Fc region.

103 gene-hIgG1 fusion proteins were produced by transient transfectionof COS-7 cells, as described in Section 6.3.1, above. Protein Aimmunoprecipitation of the COS-7 supernatants and their analysis bySDS-PAGE demonstrated, first, that the correct IgG-1 peptide was beingproduced as part of the fusion (as evidenced by the fusion's protein Aimmunoprecipitation) and, second, demonstrated substantial expression ofthe 103 gene-IgG1 fusion protein at a concentration approximately 1μg/mL of culture supernatant. Further, when the immunoprecipitatedsupernatants are analyzed and compared under reducing and non-reducingconditions, it is clear that the 103 gene-IgG1 fusion protein undergoesoligomerization, as expected, given the human IgG1 heavy chain peptidesequence present in the fusion protein. Further, the size (i.e., largerthan expected from the amino acid sequence alone) and appearance of thefusion proteins as they migrate through the gels (i.e., diffuse, ratherthan tight bands) indicate that, as expected, the fusion proteins havebeen glycosylated.

6.4. PRODUCTION AND CHARACTERIZATION OF GENE 103 TRANSGENIC ANIMALS

Described herein is the production and characterization of transgenicmice that over-express murine 103 gene products.

6.4.1. MATERIALS AND METHODS

Construction of 103 Gene Transgenic Clone:

A PCR product of the entire 103 gene sequence was used to replace theIL-10 gene in the pCIL-10 plasmid. The pCIL-10 plasmid was as describedin this Section, above. A PCR product of the entire murine long form ofthe 103 gene (Yanagisawa, K. et al., 1993, FEBS 318:83-87) codingsequence was obtained through 35 cycle-reaction using first-strand cDNAfrom a mouse TH2-type cell line, D10G4 (ATCC, MD), as template. TotalRNA was extracted from the cell line by RNAzole™ (TEL-TEST, Inc., TX).Seven micrograms RNA were used in a 20 μl first-strand cDNA synthesisreaction by Superscript Reverse Transcriptase I (GIBCO BRL, MD)following manufacturer's suggestion. Two microliters of cDNA were usedin PCR reaction. The 5′-oligo was:

(SEQ ID NO: 26) 5′-GAACACACTAGTACTATCCTGTGCCATTGCCATAGAGA-3′,and the 3′-oligo was:

(SEQ ID NO: 27) 5′-GGAATATTGGGCCCTTGGATCCCAAGTCTGCACACCTGCACT CC-3′,with compatible restriction sites SpeI at 5′-end and BamHI at 3′ end,respectively. After heat denaturation at 95° C. for 2 minutes, 3-stepcycling was performed at 45 seconds at 95° C., 45 seconds at 65° C. and60 seconds at 72° C. by Vent™ DNA polymerase (New England Biolabs,Beverly, Mass.). A final step for five minutes, at 72° C., was performedfor end-polishing. The PCR product was digested by SpeI and BamHI (NewEngland Biolabs) and ligated into the SpeI-BamHI sites of pBSKIIGHvector, containing the human growth hormone fragment from pCIL-10subcloned into the BamHI-XhoI site of pBSKII (Stratagene), which wasnamed pBS-103L-GH. The pCIL-10 fragment containing human CD2 enhancerand Pμ promoter was then ligated immediately upstream of the 103L geneof pBS-103L-GH. MaxEfficient E. coli DH5α competent cells (GIBCO BRL,MD) were used for transformation following manufacturer's suggestion.The transformants were grown in LB broth containing 0.1 μg/ml ampicillinand DNA were extracted by Qiagene Plasmid Maxi Kit (Qiagene, Calif.).Restriction analysis was performed for confirmation, and the constructwas sequenced to eliminate any possible PCR introduced mutations. Aplasmid designated pCD2-103L-GH1 was selected for production oftransgenic mice.Production of Transgenic Mice:

C3H/HEJ and FVB/NJ mice were obtained from the Jackson Laboratory (BarHarbor, Me.). Females aged 3-4 weeks were induced to ovulate byintraperitoneal injection of pregnant mare's serum (PMS) between 10 a.m.to 2 p.m., followed 46 hours later by intraperitoneal injection of humanchorionic gonadotropin (hCG). Following hCG administration, the femaleswere housed overnight with males of the same strain. The followingmorning females were examined for the presence of a copulation plug andembryos were isolated from those females with plugs, essentially asdescribed in Manipulating the Mouse Embryo (Hogan et al., eds., ColdSpring Harbor Laboratory Press, 1994).

DNA for embryo microinjection was prepared by digesting of pCD2-103L-GH1with NotI and XhoI followed by gel electrophoresis. The 9 kb and 10 kbfragments, respectively, were electrophoresed onto an NA-45 membrane(Schleicher and Schuell) by cutting a slit into the gel immediately infront of the desired band, inserting the NA-45 membrane and continuingelectrophoresis until the DNA band has been transferred to the membrane.The DNA was eluted from the membrane by incubation with 0.4 ml of 1MNaCl/0.05M arginine-free base at 65-70° C. for several hours in amicrofuge tube. The eluted DNA was extracted with phenol/chloroform andchloroform, ethanol precipitated and dissolved in 200 μl of 5 mM Tris,pH 7.5/0.1 mM EDTA. The DNA was then re-precipitated with ethanol andre-dissolved in 40 μl of 10 mM Tris, pH 7.5/0.1 mM EDTA. Prior tomicroinjection, the DNA was diluted to 1-2 μ/ml in 10 mM Tris, pH7.5/0.1 mM EDTA.

DNA was microinjected into the male pronuclei of strain C3H/HEJ orFVB/NJ embryos and injected embryos were transferred into the oviductsof pseudopregnant females essentially as described in Manipulating theMouse Embryo. The resulting offspring were analyzed for the presence oftransgene sequences by Southern blot hybridization of DNA prepared fromtail biopsies.

Southern Blot Analysis of Transgenic Alice:

Approximately ½″ piece of tail was clipped and digested in 500 μlproteinase K solution [containing 100 mM Tris HCl, pH 8.0; 5 mM EDTA, pH8.0; 0.2% SDS; 200 mM NaCl; 100 μg/ml Proteinase K (Boehringer Mannheim,Germany)] at 55° C. overnight. Digests were centrifuged for 15 minutesto remove undigested debris. Supernatants were precipitated with anequal volume of isopropanol at room temperature. Precipitates werecentrifuged for 25 minutes and pellets washed in 75% ethanol. Pelletswere air dried and resuspended in 100 μl TE; pH 8.0. Restriction digestsof tail DNA were performed as follows: 20 μl DNA solution was digestedwith 80 units BamHI (New England Biolabs) in the presence of 1 mMspermidine overnight at 37° C. Digested samples were analyzed by gelelectrophoresis using 0.8% agarose gels. Separated DNA was transferredto Hybond-N+ (Amersham, Inc.) following depurination in 0.25M HCl for 10minutes followed by 0.5 M NaOH, 1 M NaCl for 30 minutes, and then 2.5MTris-HCl (pH 7.4), 2.5M NaCl for 30 minutes. Immediately prior totransfer, gels were briefly equilibrated in a 10×SSC transfer buffer.Transfer was carried out overnight in 10×SSC by capillary action. Aftertransfer, the membrane was air dried and UV-crosslinked using aStratolinker (Stratagene, Inc.). After crosslinking, membranes wererinsed briefly in 2×SSC.

For 103 gene transgenic animals, a ³²P-radiolabelled PCR fragment of thepCD2-103L-GH construct described above was utilized. The PCR fragmentwas generated using the following primers:

5′-oligo: (SEQ ID NO: 28) 5′-GTA-AAT-CGT-CCT-GGG-GTC-TGG-3′; 3′-oligo:(SEQ ID NO: 29) 5′-CCT-TCT-GAT-AAC-ACA-AGC-ATA-AAT-C-3′.

Using these oligonucleotide primers and the pCD2-103L-GH template, PCRreactions conditions were as follows: 20 cycles of 30 seconds at 94° C.,30 seconds at 60° C. and 30 seconds at 72°, using Vent™ DNA polymerase(New England Biolabs, Beverly Mass.). Upon hybridization to mousegenomic digested with EcoRI and SpeI, the resulting probe hybridized toan endogenous 2.4 kb band and a 0.85 kb transgenic-specific band.

6.4.2. RESULTS

103 gene transgenic mice (five FVB founder lines) were producedaccording to the method described above, in Section 6.4.1. Southernhybridization analysis demonstrated the successful production of 103gene transgenic founder animals.

6.5. THE 103 GENE PRODUCT EXHIBITS A CRITICAL ROLE IN REGULATING TH2EFFECTOR CELL RESPONSES

This example presents in vivo data demonstrating that the 103 geneproduct regulates TH2 effector cell responses. In particular, theexample describes generation of a monoclonal antibody, referred toherein as 3E10 mAb, against the 103 gene product. The effect of themonoclonal antibody 3E10 mAb in an in vivo adoptive transfer model ofTH1 and TH2 immune responses is also described. Further, the examplealso describes the effect of a 103 gene product fused to an Ig tail(103/Ig fusion) in the adoptive transfer model.

Specifically, the anti 103 gene product mAb is shown to abrogate, in thein vivo adoptive transfer model, the production of IL-4, IL-5, IL-6 andIL-13, TH2 mediated lung inflammation and the associated airwayhyperresponsiveness. The data also demonstrates that the 103/Ig fusionresults in a decrease in eosinophil infiltration into and inflammationof lung airways in the model. In contrast, the 103 gene product mAbfailed to inhibit TH1-mediated lung pathology and IFN-γ secretion. Theresults therefore provide in vivo animal data indicating that the 103gene product provides a critical signal to TH2 effector cells and can beutilized as a novel target for the selective suppression of TH2 immuneresponses, e.g., in immune disorders such as asthma and allergy. Inparticular, the data presented herein demonstrates that monoclonalantibodies that specifically bind to a 103 gene product, including the3E10 mAb disclosed herein, can be used to effectively treat symptoms ofimmune disorders such as allergy and asthma in vivo.

6.5.1. MATERIALS AND METHODS

CD3/TCR Crosslinking:

Mice expressing the transgene for the DO11.10 αβ-TCR, which recognizesresidues 323-339 of chicken ovalbumin (OVA) in association with I-A^(d)(Murphy, K. M., et al., 1990, Science 250:1720-1723) were utilized.DO11.10 TCR-transgenic CD4⁺ T cells were cultured in complete RPMI 1640with OVA₃₂₃₋₃₃₉ (1 μM) and mitomycin C-treated splenocytes. For TH1phenotype development, recombinant murine Il-12 (10 ng/ml) andneutralizing anti-IL-4 mAb (11B11, 40 μg/ml, R&D Systems) were added andfor TH2 development recombinant murine IL-4 (10 ng/ml) and neutralizingpolyclonal anti-murine IL-12 (TOSH-2, 3 μg/ml, Endogen, Cambridge,Mass.) were used. Cultures were maintained for 48 hours and 5 days afterstimulation, after which time cells were harvested and purified overficoll. 1×10⁷ cells were washed and RNA extracted as described below.The remainder of the cells were stimulated on plate bound anti-CD3 inthe presence of h IL-2 (Endogen) for 48 hrs.

Anti-103 Monoclonal Antibodies:

Rat monoclonal antibodies (MAbs), including the 3E10 MAb, were generatedagainst the extracellular domain of the mouse 103 gene product. A DNAsequence containing the extracellular domain of 103 gene product wasPCR-amplified and cloned into a vector containing the CD5 signalsequence and the human IgG1 constant region. COS cells were transientlytransfected using Lipofectamine™ (GIBCO) protocol according tomanufacturer's instructions. Cells were cultured in Ultra-Low™ Ig fetalbovine serum (GIBCO) for approximately one week prior to harvest and therecombinant protein was purified by passage over a protein A column.

Lou/M rats were then immunized by subcutaneous injection of 0.5 mgpurified recombinant 103 gene product protein. Rats were boosted twicevia intraperitoneal injections at 2 week intervals with approximately300 μg purified protein. Animals were analyzed for reactivity to thefusion protein by FACS and ELISA approximately 10 days after the lastboost. Four weeks later, positive reacting animals were boosted oncemore and sacrificed 3 days later. Splenocytes were fused with SP/2myeloma cells and resulting clones were screened and selected to bespecific for the 103 gene product on the basis of their reactivityagainst 103 gene product Ig, but not CD44-Ig, and their ability to stain103 gene product COS transfectants, but not control transfectants.Pre-immune serum from non-immunized Lou/M rats was used asnegative-controls.

One of these mAbs was identified and termed 3E10.

Surface Expression of 103 Gene Product on TH2 Clones and TH2 EffectorCells:

The 3E10 mAb was labeled with digoxigenin and the number of 103-positivecells were detected by anti-digoxigenin Fab fragments (BoehringerMannheim) conjugated to Cy5.

CD4 positive, L-selectin negative cells were isolated using highgradient magnetic cell separation system MACS (Milteny; Biotec,Berg-Gladbach). Expression of 103 was analyzed on afluorescence-activated cell sorter (FACS)-calibur (Becton-Dickinson)five to seven days after restimulation with OVA peptide under theindicated polarizing conditions.

In Vitro Activation of TH2 Effector Cells:

TH2 effector cells were activated with plate-bound CD3 (1 μg/ml, 2C11)and CD28 (37.51, 4 μg/ml), Pharmingen, San Diego) and 3E110 (20 μg/ml)for 48 hours. IL-4 and IL-5 levels were measured in the supernatant byElisa.

RNA Isolation:

Total cellular RNA for RT-PCR analysis was extracted from cells usingthe Rneasy Total RNA kit (Qiagen; Chatsworth, Calif.). Poly A+ RNA (forNorthern analysis) was isolated from activated cells using FastTrackmRNA Kit (Invitrogen Corp.; San Diego, Calif.).

Northern Analysis:

1.0 μg RNA were loaded per lane for the Northern blot analysis. The 103gene probe was a 409 by RsaI fragment from the 103 gene cDNA (position1252-1661 based on the published sequence for Genbank accession numberD13695). IL-4 and beta-actin probes were purchased from Clontech, Inc.,Palo Alto, Calif. IFN-γ probe consisted of a 344 bp fragment of murineIFN-γ covering the region from position 532-876 (GenBank accessionnumber M28621). Northern blot analysis was carried out according tostandard techniques.

RT-PCR:

First strand cDNA was synthesized from equal amounts of RNA using theSuperscript Preamplification System (Life Technologies; GaithersburgMd.). PCR was performed using 25 ng of first-strand cDNA. The followinggene-specific primers were used for PCR amplification:

Gene 103: (SEQ ID NO: 30) 5′-ACGGAGGGCAGTAAATC-3′, and (SEQ ID NO: 31)5′-CAGCCAAGAAGTGAGAGC-3′; IFN-gamma: (SEQ ID NO: 32)5′-TGTTGCCGGAATCCAGCCTCAG-3′, and (SEQ ID NO: 33)5′-GTCCCCCACCCCCAGATACAACC-3′.Primers for glyceraldehyde 3-Phosphate Dehydrogenase (G3PDH) and IL-4were purchased from Clontech Laboratories (Palo Alto, Calif.).

PCR was carried out using the Advantage KlenTaq Polymerase mix (ClontechLaboratories; Palo Alto, Calif.) according to the provided protocol;annealing temperature 56° C. Samples were removed from the PCR reactionbeginning after 15 cycles and then after 5-cycle increments. Reactionsusing the minimum number of cycles to visualize the gene of interest,were loaded onto 1.5% agarose gels for analysis.

TH Recipient Mice:

TH1 and TH2 subsets were generated as described above. Mice wereinjected with 2×10⁶ TH2 cells intravenously into recipient BALB/c mice.Twenty four hours later, mice were exposed daily to an aerosol of OVA(50 mg/ml) (Grade V, Sigma, St. Louis) for 20 min for 2 consecutivedays. Control mice were either injected with TH2 cells and exposed to anaerosol of PBS or were exposed to OVA in the absence of cell transfer.Mice were sacrificed 24 hrs after the last aeroallergen challenge. Onehour prior to allergen exposure, mice were injected with either 20 μg or100 μg of 3E10 mAb, recombinant 103 gene product-IgG fusion protein, or100 μg of rat IgG1 (Sigma, St. Louis) as the appropriate isotypecontrol. Twenty fours after the last challenge, the trachea wascannulated and a bronchoalveolar lavage performed with 4×0.3 ml aliquotsof PBS (Gonazlo, J. A., et al., 1996, Immunity 4:1-14). Cytokine levelsin the lavage fluid were measured by ELISA (PharMingen, San Diego).

Flow Cytometry Analysis of TH Clones:

AE7 (TH1), Dorris (TH1), DAX (TH2) and D10-G4 (TH2) clones were analyzedfor the expression of gene 103 protein using fluorescence activated cellsorting (FACS). Cells were stimulated with appropriate antigen andcultured for approximately 3 days prior to analysis. Pre-immune serumwas prepared for unimmunized Lou/M rats.

50 μl of 3E10 culture supernatant (or 1 μg purified 3E10 protein) wasapplied 1×10⁶ cells. After rinsing, cells were contacted with goatanti-rat antibody conjugated with PE (R-phycoerythrin) fluorescent dye.After a final rinse, cell analysis was carried out on a FACS Vantage(Becton Dickenson).

103/Ig Fusion Protein:

The 103/Ig fusion proteins were generated as discussed, above, in theExample presented in Section 6.3.

Cell Preparation and Polarization:

Spleens from DO11.11 OVA aβ TCR mice were removed and CD4+ T cells werepurified by negative selection. Cells were plated at a density of1×10⁶/ml in 75 mm² flasks and stimulated with 10 μg/ml OVA peptide andmitomycin C treated splenocytes at a ratio of 1:1 CD4: APC. Cells werecultured in the presence of IL-4 (20 ng/ml) and anti-IL-12 (3 μg/ml) forTH2 polarization, or IL-12 (20 ng/ml) and anti-IL-4 (40 μg/ml) for TH1polarization. This procedure was repeated for 3 rounds of polarization.Cells were then harvested, dead cells removed by density centrifugation.TH1 and TH2 cells were then incubated at 1×10⁶/ml for 48 hrs in IL-2alone (10 ng/ml).

Adoptive Transfer Model:

2×10⁶ cells were injected intravenously via the tail vein into recipienttransgenic mice. Twenty four hours later, mice were exposed daily to anaerosol of OVA (50 mg/ml) antigen (Grade V, Sigma, St. Louis) for 20minutes. Control mice were exposed to an aerosol of PBS alone. Mice weresacrificed on days 3, 5 and 7. In separate experiments, mice received 20μg/mouse i.v. of either 3E10 mAb or the 103 Ig fusion protein. Controlmice were injected with 20 μg of either rat or human Ig as theappropriate isotype control. This procedure was repeated for twoconsecutive days.

24 hours after the last challenge, mice were anaesthetized with 0.3 mlof 14% urethane i.p. and the trachea cannulated. A bronchoalveolarlavage (BAL) was performed by injecting 0.3 ml of PBS into the lungs.The fluid was then withdrawn and stored on ice. This procedure wasrepeated a total of 4 times. The cell suspension was then centrifuged (5mins, 1500 rpm, 4° C.) and the supernatant removed and frozen at −20° C.The cell pellet was then resuspended in 1 ml of PBS and total cellcounts were obtained. Cytospin® (Shandon, Inc., preparations were thenprepared and stained with Diff-Quik (Baxter Corporation). A total of 200cells were then counted differentially using standard morphologicalcriteria. Cytokine levels were measured in the BAL fluid by ELISA(Pharmingen, San Diego).

Active Immunization Protocol and IgE Measurement:

Male BALB/c mice (15-20 g) were immunized intraperitoneally with 7.5 μgof OVA and 1.5 mg Al(OH)3 in saline on Day 0 and Day 7. On day 14 andDay 21 the mice were challenged with aerosolized OVA (10 mg/ml) for 1hours. Control mice were challenged with PBS instead of OVA. One hourprior to each allergen sensitization and challenge, the mice wereinjected with 100 μg of 3E10 mAb or 100 μg of rat IgG1 (Sigma, St.Louis). Twenty-four hours following the second allergen challenge a BALwas performed and IL-5 levels in the BAL fluid determined. Serum OVAspecific IgE was determined by specific ELISA.

Airway Responsiveness:

Airway responsiveness was measured in TH2 recipient mice, 24 hours afterthe last aerosol challenge by recording respiratory pressure curves bywhole body plethysmography (Hamelmamn, J. E., 1997, Am. J. Respir. Crit.Care Med. 156:766-775); Buxco®, EMKA Technologies, Paris, France) inresponse to inhaled methacholine (Aldrich-Chemie, Steinhein, Germany) ata concentration of 2.5 to 25 mg/ml for 1 minute. This method allowedmeasurements of spontaneous breathing in a non-restrained mouse. Airwayresponsiveness was expressed in enhanced pause (Penh), a calculatedvalue, which correlates with measurement of airway resistance, impedanceand intrapleural pressure in the same mouse. Penh=(Te/TR1)×Pef/Pif(Te=expiration time, Tr=relaxation time, Pef=peak expiratory flow,Pif=peak inspiratory flow) (Hamelmamn, J. E., 1997, Am. J. Respir. Crit.Care Med. 156:766-775).

Lung Histology:

Following the BAL analysis, lungs were inflated with 0.6 ml of a mixtureof OCT compound (Tissue-kek®; Miles Inc., Elkhart, Ind.) and 20% sucrose(Sigma, St. Louis, Mich.) at a ratio of 1:1. The lungs were thenremoved, snap-frozen and 8-10 μm cryosections fixed in methanol at 20°C. for 2.5 minutes. Slides were stained with haematoxylin and eosin(Fluka Chemika, Buchs, Switzerland).

In Situ Hybridization:

Recipient Balb/C mice were injected intervenously with 2×10⁶ TH1 or TH2cells generated as described above. Twenty-four hours later, mice wereexposed to an aerosol of OVA (50 mg/ml; Grade V, Sigma) for 20 minutesfor two consecutive days. Mice were sacrificed 24 hours after the lastaeroallergen challenge. Lungs were removed and snap frozen for in situhybridization. A 35-mer antisense oligonucleotide against 3′-UTR 103gene sequence was synthesized and end-labeled as follows: 100 pmol oligowas incubated for 15 minutes at 37° C. with 10 nmol dATP (Promega), 40μmol biotin-dUTP, 1× terminal transferase buffer, 5 mM CoCl₂, 50 unitstransferase (Boehringer-Mannheim, Germany). Formalin fixed 5 μm tissuesections were hybridized for 16-18 hours. Control slides were hybridizedwith probe mix containing 50-fold excess unlabelled oligo. Hybridizedprobe was detected with a biotinyl tyramide amplification method(GenPoint, Dako) and visualized by addition of AEC substrate kit(Vector) for five minutes.

6.5.2. RESULTS

RT-PCR analysis performed herein demonstrates that the 103 gene isinduced only upon CD3/TCR crosslinking during differentiation of TH0 toTH2, but not TH1 effector cells. The RT-PCR analysis was confirmed byNorthern analysis. These data corroborate the results presented in theExample of Section 6.2, above.

To further investigate the expression and role of the 103 gene productin TH cells, a monoclonal antibody (3E10 mAb) directed against theextracellular domain of the 103 gene product was prepared andcharacterized.

Flow cytometry data is presented in FIG. 17 which demonstrates that the3E10 mAb recognizes and binds to representative clones of the TH2 cellsubpopulation (D10.G4; DAX), but not clones of the TH1 subtype (AE7;Dorris). For these experiments, cells were contacted with 3E10 mAb,preimmune serum (negative control) or a second antiserum (positivecontrol; referred to as “αTH1 serum” for AE7 and Dorris, and “rat α103serum” for D10.G4 and DAX). In contrast, this mAb failed to recognizeresting or activated CD4+ (L-selectin), CD8+, B cells or macrophagecells.

When TH1 cells (AE7, Dorris) were analyzed, the peaks for 3E10 MAb andthe negative preimmune serum exhibited the same very low level ofstaining as the negative control preimmune serum. No detectable 103 geneproduct is present, therefore, on the surface of the TH1 cells. Incontrast, with TH2 cells (D10.G4, DAX), the 3E10 MAb peak shiftedsignificantly to the right, demonstrating the presence of 103 geneproduct on the TH2 cell surface. It is noted that for each cloneanalyzed, the positive control peak is shifted well to the right ofbackground levels, as expected.

In addition to the TH2-specific expression pattern observed inestablished TH clones as discussed above, 3E10 mAb staining and flowcytometry analysis was utilized to successfully demonstrate that 103expression dramatically increases when freshly isolated TH cells arecultured under Conditions that induce TH2 cell polarization, with theexpression being dependent on the degree of differentiation of the TH2phenotype. Such an increase was not observed naive CD4+ (L-selectionnegative) under TH1 cell polarization conditions (i.e., TH1 effectorcells derived from the TH precursor cells).

As shown in FIG. 18, pretreatment of TH2 recipient mice with 3E10 mAbinhibited the secretion of IL-4, IL-5, IL-6 and IL-13 by greater than90%. In particular, analysis of the cytokine profile in the BAL revealedhigh levels of IL-4, IL-5, IL-6, IL-10 and IL-13 in TH2 recipient OVAchallenged mice (closed bars). There was no detectable TH2 cytokines inthe BAL fluid of mice that received TH2 cells and were not exposed toovalbumin. Pretreatment with 3E10 mAb resulted in a dramatic reductionin IL-4, IL-5, IL-6 and IL-13, but had no effect on IL-10 levels in theBAL (open bars). OVA challenge of TH1 recipient mice resulted in highlevels of IFN-γ in the BAL fluid (closed bars) that was not inhibited by3E10 mAb (open bars).

These data show that the 103 gene is differentially expressed in aTH2-specific manner, thereby corroborating the results presented in theExample of Section 6.1, above. In addition, the data demonstrate thefeasibility of using antibodies to separate TH2 subpopulation cells awayfrom other cell types, thereby modulating a TH cell subpopulation bychanging the number of cells belonging to one TH cell subpopulationrelative to that of another TH cell subpopulation.

An in vivo TH1 and TH2 adoptive transfer model (Cohn, L. et al., 1997,J. Exp. Med. 186:1737-1747) was used to address the role of the 103 geneproduct in TH cells. In this adoptive transfer animal model,aeroallergen provocation of TH1 or TH2 recipient mice results in THeffector cell migration to the airways and is associated with an intenseneutrophilic (TH1) and eosinophilic (TH2) lung mucosal inflammatoryresponse. The model represents an accepted animal model for asthma, aTH2-like disorder. In situ hybridization revealed a marked upregulationof 103 gene mRNA positive cells in lungs from allergen or PBS provokedTH2 recipient mice. In marked contrast there was no detectable 103 genemRNA expression in lungs obtained from either OVA or PBS provoked TH1recipient mice.

The animal model was used to investigate whether neutralization of the103 gene product in vivo also abrogated TH2-mediated pathology. Allergenprovocation of mice which had received TH2 cells and control rat Igresulted in infiltration of lymphocytes and eosinophilic inflammation ofthe airways. In vivo administration of 3E10 mAb markedly suppressed thedevelopment of eosinophilic inflammation of the airways. In particular,eosinophilic inflammation was assessed, first, by histological analysisof the airway tissue. Second, an analysis of the cellular composition ofthe bronchoalveolar lavage fluid (BAL) was performed (FIGS. 19A-B). Nosignificant reduction in the number of antigen specific TH2 cells thatmigrated into the airway interstitium after allergen challenge wasobserved.

In marked contrast to the effects on TH2 immune responses, 3E10 mAbtreatment did not suppress IFN-γ secretion or neutrophilic lunginflammation induced by allergen challenge of TH1 recipient mice. It isalso of interest to note that the anti-103 gene product mAb failed toinhibit IL-10 secretion, a cytokine that has been shown to suppresseosinophil infiltration and prevent IgE mediated mast cell activation.

TH2 mediated lung mucosal eosinophilic inflammation is associated withheightened airway responsiveness to non specific stimuli and is acharacteristic feature of bronchial asthma (Ohashi, Y. et al., 1992, Am.Resp. Dis. 145:1469-76). To determine whether the 103 gene product isinvolved in this physiological consequence of allergen exposure, thedegree of airway constriction induced by the methacholine inhalation wasassessed using whole body plethysmography.

3E10 mAb treatment was, indeed, demonstrated to attenuate allergeninduced heightened airway responsiveness. In particular, 3E10 mAbtreatment suppressed the development of airway hyperresponsivenessinduced by OVA challenge in TH2 recipient mice (FIG. 20). TH effects oftreatment with 3E10 mAb were comparable to those previously reportedusing anti-IL-5 mAbs Wang, L. M., 1992, EMBO 11:4899-4908 and anti-B7-2mAbs (Tsuyuki, S. et al., 1997, J. Exp. Med. 185:1671-1679).

The role played by 103 gene in lung inflammation was also investigatedin an active immunization model where mice were injected systemicallywith antigen and adjuvant prior to two repeated allergen provocations.As summarized in FIGS. 21A-B, administration of either 3E10 mAb or103/Ig fusion results in a significant reduction in eosinophilicinflammation, as well as a significant reduction in IL-4 and IL-5cytokine levels in the lung, which represent cytokine hallmarks ofactivated TH2 cell subpopulations. In addition, 3E10 mAb attenuated theinduction of OVA specific IgE in the serum of the active immunizationmodel. Still further, an approximate 60% reduction in airwayhyperresponsiveness was observed after repeated aerosol challenge afteractive immunization.

Further, the level of interferon gamma was measured, which represents ahallmark of TH1 cell subpopulation activation, and an increase in itslevel was detected. This indicates the presence of a relative increasein TH1 cell subpopulation responses.

In addition, animals were treated with a soluble fusion proteincontaining the extracellular domain of the 103 gene product fused to anIg tail (103/Ig fusion). Administration of the 103/Ig fusion results insignificant decrease in hallmark symptoms of asthma. As summarized inFIG. 21B, such administration results in animals that exhibit a decreasein eosinophil infiltration into lung airways (this was assessed by bothBAL and histological examination). Likewise, administration of the103/Ig fusion resulted in a 50% attenuation in the degree ofeosinophilic inflammation of airways.

Thus, the inhibition of 103 gene function appears to modulate TH cellsubpopulations by decreasing the level and/or activity of TH2 cellswhile bringing about a relative increase in the level and/or activity ofTH1 cells.

To determine whether signalling through the 103 gene product directlymodifies cytokine production, TH2 effector cells were activated withplate bound CD3 and CD28. Under conditions where Fc crosslinkingoccurred, 3E10 mAb augmented IL-4 and IL-5 secretion in the absence ofenhanced proliferation (FIG. 22). In contrast, CD3/CD28 stimulation ofTH1 cells in the presence of plate bound 3E10 mAb failed to modify IFN-γsecretion. These results indicate that ligation of the 103 gene productin conjunction with signals delivered through the CD3/CD28 complextogether with CD28 mediated co-stimulation provide a novel costimulatorysignal specific for TH2 effector cells.

Recently, GATA-3 have been shown to be preferentially expressed in TH2cells and suggested to play an important role in TH2 differentiation(Zheng, W.-P. & Flavell, R. A., 1997, Cell 89:587-596): Unlike GATA-3,however, the 103 gene product is induced upon CD3/TCR mediatedactivation and not during TH2 differentiation from TH0 cells. GATA-3 maybe involved in TH differentiation, while the 103 gene product may bemore involved during activation of TH2 effector cells. Further, the 103gene promoter in murine mast cells contains a GATA-3 consensus bindingsequence (Gachter, T. et al., 1996, J. Biol. Chem. 271:124-129),indicating that GATA-3 may be involved in the TH2 specific expression ofthe 103 gene.

In summary, these results provide both in vitro characterization of 103gene expression and the 103 gene product, as well as in vivo animal dataindicating that the 103 gene product provides a critical signal to TH2effector cells and represents a critical regulatory molecule for bothcellular and humoral allergic inflammation. These data indicate that the103 gene and/or gene product can be utilized as a novel target for theselective suppression of TH2 immune responses. For example, the datapresented herein demonstrates that monoclonal antibodies thatspecifically bind to a 103 gene product, including the 3E10 mAbdisclosed herein, can be used to effectively treat symptoms of disorderssuch as allergy and asthma in vivo.

6.6. THE 103 GENE PRODUCT IS EXPRESSED IN HUMAN MAST CELLS

The example provided herein presents data demonstrating that the 103gene product is expressed in mammalian mast cells. First, Northernanalysis showed high levels of 103 gene expression in a human mast cellline. FAC staining of the human mast cell line demonstrated binding ofanti-103 monoclonal antibodies, confirming that the 103 gene product is,indeed, expressed in human mast cells. It is noted that theseantibodies, the production of which is also described hereinbelow, arespecific for human, but not murine, 103 gene products.

6.6.1. MATERIALS AND METHODS

103 Fusion Proteins:

A human 103 IgG1 Fc fusion protein was constructed and utilized togenerate monoclonal antibodies directed against the extracellular domainof the human 103 gene product. Specifically, this fusion proteincontained, from amino- to carboxy-terminus, a CD5 signal sequence(CD5ss), glycine and threonine amino acid residues (a Kpn1 site), theextracellular domain (i.e., amino acid residues 18 to 323) of the human103 gene product depicted in FIG. 5B (SEQ ID NO:8) and human IgG1 Fc.The vector encoding this fusion protein was transfected into mammalian293T cells using LipofectAMINE® (GIBCO BRL, Md.) following themanufacturer's recommended protocol. Supernatants were harvested 3 and 7days, respectively, after transfection, and fusion protein was purifiedwith a Protein G affinity column (Pierce, Inc., IL.) according to themanufacturer's recommended protocol.

A second Fc fusion protein of the human 103 gene product wasconstructed, according to the techniques described in Section 6.3 above,to bind ELISA plates for screening. In particular, the fusion proteincontained, from amino- to carboxy-terminus, a T075 signal sequence(T075ss; the signal sequence of TANGO 75 in PCT Publication No. WO99/15663, filed Apr. 1, 1999) plus QR residues, amino acid residues20-323 of the human 103 gene product (FIG. 5B; SEQ ID NO: 8) plus alinker amino acid sequence (Ala-Ala-Ala-Asp-Pro) and a human IgG1constant region.

A fusion protein of the mouse 103 gene product was also constructed andutilized that contained, from amino- to carboxy-terminus, a CD5 signalsequence plus GT residues, residues 24 (Thr) to 328 (Pro) of the mouse103 gene product (FIG. 4B; SEQ ID NO:6), and human IgG1 constant region.

Control fusion proteins were also utilized which contained unrelatedproteins, T001, a chemokine (referred to as TANGO 1 in PCT PublicationNo. WO 97/4224, filed Nov. 13, 1997), or T075, a tumor necrosis familyreceptor (referred as TANGO 75 in PCT Publication No. WO 99/15663, filedApr. 1, 1999), fused with a human IgG1 constant region.

The human IgG1 sequence was as described in Aruffo et al., 1990, Cell61:1303.

Generation of Anti-103 Monoclonal Antibodies:

Monoclonal antibodies were generated in BALB/c mice against theextracellular domain (amino acid residues 18-323) of the human 103 geneproduct depicted in FIG. 5B (SEQ ID NO: 8) utilizing the above-describedhuman 103 IgG1 Fc fusion protein (see Section 5.6, above) for monoclonalantibody selection and purification. Specifically, BALB/c mice wereimmunized with the fusion protein according to standard protocols andspleen cells from a mouse that showed positive reactivity to the 103fusion protein were fused with SP2/0 myeloma cells using standardpolyethylene glycol (PEG) techniques (see, e.g., Harlow and Lane, 1988,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.; Current Protocols in Immunology, 1992,Coligan, J. E. et al., Eds., John Wiley & Sons, NY).

The resulting hybridoma cell lines were screened to select clones whichsecreted antibodies that bound specifically to the human 103 IgG1 Fcfusion protein but not to control Fc fustion by ELISA and Biacore(BIAcore, Inc.; Uppsala, Sweden) as described in Fagerstam et al., 1992,J. of Chromatography 597:397-410 and in Kretschmann & Raether, 1968, Z.Naturforschung Teil. A. 23:2135. These assays used the above-describedplate bound human 103-IgG1 Fc fusion protein and control fusion proteinsto select monoclonal antibodies that specifically bound to a human 103gene product but did not bind to mouse 103-gene product Fc fusionprotein or to a control Fc fusion protein.

Six cell lines were selected and cloned using ClonalCell™-HY Medium D(StemCell Technologies, Inc., Vancouver, BC) according to themanufacturer's recommendations and purified from DMEM 3% FCS (Ultra-lowIgG, Gibco BRL) supplemented with Penicillin/Streptomycin andL-glutamine. The antibodies were purified by Protein A affinitychromatograph, using standard protocols, and affinities were analyzed byBiacore as described above. The isotypes of the purified monoclonalantibodies were determined using a commercial isotyping kit (Pharmingen,San Diego) following the manufacturer's recommended protocol.

Northern Blots:

Northern procedures performed in the experiments described in thisexample were performed as described, above, in Section 6.1.

Flow Cytometry Analysis of Mast Cell Lines:

Cells were analyzed for the expression of gene 103 protein usingfluorescence activated cells sorting (FACS) according to standardmethods described in Section 6.5, above using anti-mouse IgFITCsecondary antibodies.

6.6.2. RESULTS

Northern blot analysis of multiple cells lines showed high levels of the103 gene in a human mast cell line. Expression of the 103 gene productin this cell line was verified using monoclonal antibodies raisedagainst an Fc fusion protein of the human 103 gene product, as describedin Subsection 6.5.1, above.

Specifically, six hybridoma cell lines, referred to herein as M15 3F7.3,M15 9F11.1, M15 2O3.1, M15 5A16.1, M15 10F7.1 and M15 1B4.1, wereselected, as described in Subsection 6.6.1, above, which producedmonoclonal antibodies that bind specifically to the extracellular domainof the human 103 gene product but do not bind to a mouse 103 geneproduct or to control fusion proteins (i.e., fusion proteins of T001 orT075, see Subsection 6.6.1, above). The isotypes of the monoclonalantibodies produced by these cell lines were determined, and are asfollows: M15 3F7.3: IgG1 Kappa; M15 9F11.1: IgG1 Kappa; M15 2O3.1; IgG2aKappa; M15 5A16.1: IgG1 Kappa; M15 10F7.1: IgG2a Kappa; and M15 1B4.1:IgG2a Kappa. The cell lines were deposited with the American TypeCulture Collection (ATCC™) in compliance with the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thepurpose of Patent Procedure (see Section 7, below). Such monoclonalantibodies, as well as hybridoma cell lines which produce suchmonoclonal antibodies, are considered to be part of the presentinvention.

FACS staining of the human mast cell line with the six monoclonalantibodies showed positive staining with four of the six antibodies (M153F7.3, M15 2O3.1, M15 10F7.1 and M15 1B4.1) compared to isotypecontrols. The positive FAC staining with these antibodies was alsodemonstrated to be specifically blocked with an excess of the human103-IgG1 Fc fusion protein. However, positive FAC staining was notblocked with control Fc fusion proteins.

Interestingly, no positive FACS staining of the human mast cell line wasobserved with the monoclonal antibodies M15 5A16.1 and M15 9F11.1. Theseantibodies, therefore, did not recognize the 103 gene product expressedon the human mast cell line, and may recognize a different epitope ofthe human 103 gene product than do the other four monoclonal antibodiesstudied.

In summary, these results provide evidence that the 103 gene product isexpressed in a human mast cell line. Accordingly, the 103 gene, its geneproduct, and compositions derived therefrom (e.g., antibodies and othercompounds which bind to and/or modulate the expression or activity ofthe 103 gene or its gene product) may be used not only in the treatmentand regulation of immune disorders such as allergy and asthma (i.e., inimmune disorders associated with an abnormal or inappropriate TH2 orTH2-like immune response), but may also be used in the treatment andregulation of mast cell related disorders. Such mast cell relateddisorders include, but are not limited to, atherosclerosis (see, e.g.,Metzler and Xu, 1997, Int. Arch. Allergy Immunol. 114:10-14), myocardialischemia/reperfusion (see, e.g., Frangogiannis et al., 1998, Circulation98:699-710), mastocytosis (e.g., cutaneous mastocytosis and systemicmastocytosis), and interstitial cystitis (IC).

6.7. THE 103 GENE PRODUCT PLAYS A CRUCIAL ROLE IN TH2-MEDIATED IMMUNERESPONSES

The example presented herein substantiates the above-described findingsthat the 103 gene product is, indeed, a gene product differentiallyexpressed in TH2 cells and plays an important role in TH2 mediatedimmune responses and can therefore serve as a novel target in thesuppression of such responses. Specifically, the data presented hereinshows that, not only is the 103 gene product differentially expressed inTH2 and not in TH1 cells, but also that the 103 gene product delivers animportant signal instructing naive (i.e., undifferentiated) TH cells toswitch to a TH2-like pattern of cytokine production.

6.7.1. MATERIALS AND METHODS

Generation of Fusion Proteins and Monoclonal Antibodies:

A 103 gene IgG1 fusion protein (103-Ig) was prepared according to themethods described in Section 6.3 above. In addition, a DNA sequencereferred to as H1 was PCR-amplified and cloned into the identical vectorusing the same methods. H1 contained the extracellular domain of an Igsuperfamily member. The H1-Ig fusion protein failed to bind to either T,B or dendritic cells. Further, H1-Ig was not detectable by PCR analysisin either resting or activated TH1 or TH2 cells. Thus, the H1-Ig fusionprotein was used as an control reagent in the below-describedexperiments.

The anti-103 mAb 3E10 mAB was generated and used as described in Section6.5, above.

CD3/TCR Crosslinking:

Mice expressing the transgene for the DO11.10 αβ-TCR, which recognizesresidues 323-339 of chicken ovalbumin (OVA) in association with I-A^(d)(Murphy, K. M., et al., 1990, Science 250:1720-1723) were utilized.Naive TCR-transgenic CD4⁺ T cells were isolated as described by Lohninget al. (1998, Proc. Natl. acad. Sci. U.S.A. 95:6930-6935) and culturedin complete RPMI 1640 with OVA₃₂₃₋₃₃₉ (1 μM) and mitomycin C-treatedsplenocytes in a ratio of 1:5. For TH1 phenotype development,recombinant murine IL-12 (10 ng/ml) and neutralizing anti-IL-4 mAb(11B11, 40 μg/ml, R&D Systems) were added and for TH2 developmentrecombinant murine IL-4 (10 ng/ml) and neutralizing polyclonalanti-murine IL-12 (TOSH-2, 3 μg/ml, Endogen, Cambridge, Mass.) wereused. After 5-7 days cells were washed and restimulated up to 3 timesunder identical polarizing conditions. Cells were stained after 5-7 dayswith digoxigenin labeled 3E10 and the number of 103 positive cellsdetected by anti-digoxigenin Fab fragments (Boehringer Mannheim)conjugated to Phycoerythrin. Expression of 103 gene product was analyzedon a fluorescence-activated cell sorter (FACS)-Calibur(Becton-Dickinson). To determine the cytokine profile at each timepoint, cells were washed and viable CD4⁺ cells were isolated over aficoll gradient and activated in a 96 well (2×10⁵ per well) plate for 24hours using platebound CD3 (2C11, 10 μg/mL, Pharmingen, San Diego). IL-4and IFN-γ levels were measured in the supernatant by ELISA (Endogen,Cambridge Mass.).

In Vitro Differentiation of Effector Cells:

CD4⁺ T cells from DO11.10 αβ-TCR mice were activated as described abovein the absence of exogenous cytokines (i.e., in “neutral” conditions) orin the presence of IL-12 or IL-4, together with 103-Ig (100 μg/mL) orhuman-Ig was the appropriate isotype control Cells were washed andreplated in 96 well plates (5×10⁴ per well) together with 1×10⁵splenocytes per well, and restimulated with OVA peptide and cytokinesmeasured 48 hours later. To determine the effect of 103-Ig in effectorcells, TH1 and TH2 cells were reactivated with OVA peptide in thepresence of either 103-Ig of H1-Ig. In some experiments H1-Ig was usedas a second control reagent for the specificity of 103-Ig.

In Vivo Measurement of TH1 or TH2 Immune Responses:

TH1 and TH2 recipient mice were generated as described in Section 6.4above. However, a second series of experiments were also performed using103-Ig (100 μg i.v.) or human Ig as the appropriate isotype control.Cytospin preparations were prepared and stained with Giemsa reagent. Atotal of 200 cells was differentially counted. Lungs were then removed,inflated with 10% neutral buffered formalin, and paraffin-embedded. Fourmicron sections were stained for cyanide-resistant peroxidase andcounterstained with Haematoxylin using standard techniques. Airwayinflammation was determined by semiquantitative scoring using anarbitrary system wherein a score of +1 represents one small foci ofcells and +5 indicates widespread infiltrates. All scoring was performedby a clinical investigator unaware of the treatment.

Active Immunization Protocol and IgE Measurement:

Mice were immunized and serum OVA specific IgE levels were measured asdescribed in Section 6.4.1, above.

6.7.2. RESULTS

To further investigate the differential expression pattern of the 103gene, 103 gene expression was determined by flow cytometry onsplenocytes, purified naive CD4+ cells (CD4⁺/CD62L+) and TH2 and TH1effector cell populations (FIGS. 23A-23D, respectively). Briefly, TH1phenotype development was stimulated by culturing naive CD4⁺ T cells inthe presence of recombinant murine IL-12 and in neutralizing anti-IL-4antibody, as described, above, in Section 6.7.1. TH2 phenotypedevelopment was stimulated by culturing the naive CD4⁺ T cells inrecombinant murine IL-4 and neutralizing anti-IL-12 antibody. 103 geneexpression was determined after primary, secondary, and tertiaryrestimulation of these cells.

The 3E10 monoclonal antibody failed to detect 103 gene product on naivecells or on TH1 cells, but recognized and bound to 103 gene product onTH2 cells. Further, stimulating naive T cells in TH2 polarizingconditions (i.e., incubating in IL-4 and anti-IL-12) actually increasedthe percentage of cells expressing the 103 gene product (from 5.2% afterprimary restimulation to 41% after tertiary restimulation; FIG. 23C).This increase in 103 gene product expression also correlated with anenhanced secretion of IL-4, a cytokine secreted by TH2 and TH2-likecells and associated with TH2 and TH2-like activity. Thus, the resultsconfirm that a majority of cells exhibiting TH2 and/or TH2-like activityalso express the 103 gene product. Thus, the data show that the 103 geneproduct is a useful surface marker for identifying cells exhibiting TH2or TH2-like activity (e.g., secretion of cytokines such as IL-4 and/orIL-5) both in vivo and in vitro. The 103 gene product can therefore beused, e.g., to modulate numbers of TH2 and/or TH2-like cells present ina population.

Cells were also incubated in the presence of 103-Ig fusion protein todetermine the role of the 103 gene product as a signaling molecule.Naive T cells that were not incubated in the presence of exogenouscytokines or in the presence of 103-Ig acquired the capacity to secretehigh levels of IL-4 (≈1700 ng/mL) and IL-5 (≈1600 ng/mL), and secretedlow levels of IFN-γ (≈300 pg/mL). However, when incubated under TH2polarizing conditions and with 103-Ig, TH2 cytokine production (i.e.,IL-4 and IL-5) was reduced while a modest, but reproducible, increase insecretion of the TH1 cytokine IFN-γ (≈1500 pg/mL) was observed (FIG. 24,top). In contrast, incubation in the presence of 103-Ig failed to modifyIFN-γ production in cells incubated under TH1 polarizing conditions(FIG. 24, bottom). Thus, administration of the 103-Ig fusion proteineffectively blocks 103-mediated signaling, e.g., by competing withendogenous 103 gene product for its ligand, effectively inhibiting TH2and/or TH2-like, but not TH1 or TH1-like, activity.

These results are similar to data generated using fusion proteins toinhibit CD28/B7 interactions (Sedar et al., 1994, J. Exp. Med.179:299-304) or B7 deficient antigen presenting cells (Schweitzer andSharpe, 1998, J. Immunol. 161:2762-2771). However, the results alsodiffer from previous findings in that the inhibition of the 103 geneproduct results in skewing of the immune response from a TH2 to a TH1phenotype. Further, whereas the absence of CD28 costimulation results inan attenuation of IFN-γ and IL-4 secretion when cells are cultured undereither TH1 or TH2 polarizing conditions, inhibition of 103 genesignaling selectively inhibits cytokine secretion from TH2 cells withoutmodifying IFN-γ secretion from TH1 cells. Thus, the 103 gene productappears to deliver an important signal instruction naive cells todifferentiate to TH2 cytokine production.

The requirement of 103 signaling or activation of effector cells wasfurther examined by activating separate TH1 and TH2 effector cellpopulation with a peptide and antigen presenting cells in the presenceof different concentrations of the 103-Ig fusion protein. Under theseconditions, blockage of 103 signaling reduced cytokine production in TH2effector cells, but not in TH1 effector cells, in a dose dependentmanner (FIG. 25). The specificity of the 103-Ig was verified inidentical experiments using the control H1-Ig fusion protein.

The results are in marked contrast to the recent data generated using B7deficient antigen producing cells (Schweitzer and Sharpe, 1998, J.Immunol. 161:2762-2771). Specifically, the previous data demonstratedthat cytokine production from TH1 and TH2 effector cells, respectfully,is largely dependent of CD28/B7 mediated costimulation. However, thedata presented here suggests that signaling through 103 accounts, atleast in part, for CD28/B7 independent activation of TH2 but not TH1cells.

The contribution of the 103 gene and its gene product to a nascentTH2-dominated response was also investigated in vivo. Briefly, mice wereimmunized systemically with antigen in adjuvant prior to allergenprovocation, as described in Section 6.5 above, and both cellular andhumoral responses were evaluated. The results are shown in FIG. 26A-26C.Anti-103 mAb effectively inhibited allergen induced lung eosinophilicinflammation, IL-5 production and the induction of OVA specific IgE,demonstrating 103 gene product is a critical regulatory molecule forboth cellular and humoral allergic inflammation in vivo.

The data demonstrate that the 103 gene product is not only a usefulmarker for identifying and detecting TH2 cells, but also plays a crucialrole in the differentiation and activation of TH2, but not TH1, cells.In particular, this conclusion is supported not only by the in vitrodata presented in this section, but also by in vivo data presented inboth this section and in Section 6.5, above. These data show thatinhibition of 103 signaling attenuates TH2 mediated immune responseswithout affecting TH1 mediated responses. Thus, the 103 gene isapparently an important regulator for a number of key events in bothinnate and adaptive immunity and, as such, is an important target forthe therapeutic and diagnostic methods and compositions describedherein.

6.8. GENERATION OF HUMAN ANTIBODIES TO THE HUMAN 103 GENE PRODUCT

The Examples presented in the previous sections (e.g., in Sections 6.5and 6.6) describe the production of exemplary mouse monoclonalantibodies that specifically bind to a murine and a human 103 geneproduct, respectively. The Example presented in this Section presentsdata demonstrating the production of exemplary human antibodies thatspecifically bind to a human 103 gene product. Such human antibodies areparticularly useful in the methods and compositions of the presentinvention including, in particular, those embodiments of the inventionthat comprise administering antibodies to a human subject.

6.8.1. MATERIALS AND METHODS

103 Fusion Proteins:

Human 103 IgG1 Fc fusion protein, prepared as described in Section 6.6,above, was used to generate monoclonal antibodies directed against theextracellular domain of the human 103 gene product. A second Fc fusionprotein of the human 103 gene product was also constructed to bind toELISA plates for screening. In particular, this fusion protein wasidentical to the plate bound human 103 fusion protein described inSection 6.6.1, above, except that the fusion protein contained a leadersequence from the human death receptor (DR6), T075ss, instead of CD5ss,plus amino acid residues QR, amino acid residues 20-323 of the human 103gene product and a murine IgG1 constant region rather than a human IgG1constant region.

A third human 103 fusion protein was also constructed for expression onthe surface of transfected cells. This fusion protein contained, fromamino- to carboxy-terminus, the leader sequence of human death receptor(DR6), amino acid residues 20 to 323 of the human 103 gene productdepicted in FIG. 5B (SEQ ID NO:7), a His tag and the C terminal signalsequence from human placental alkaline phosphatase. The vector encodingthis fusion protein was transfected into mammalian 293T cells usingstandard protocols (see, e.g., Section 6.5.1, above). For controls,mammalian 293 T cells were also transfected with a vector that consistedof the human DR6 leader sequence, a His tag and the human placentalalkaline phosphatase C terminal signal sequence, but did not containsequence for a 103 gene product.

Generation of Human Antibodies:

Monoclonal antibodies were generated in xenomice expressing human IgG2(Abgenix, Inc., 7601 Dumbarton Circle, Fremont, Calif. 94555; B6×129transgenic produced by microinjection of B6 embryos with 129 ES cells)against the extracellular domain (amino acid residues 18-323) of thehuman 103 gene product depicted in FIG. 5B (SEQ ID NO:7) utilizing theabove-described human 103 IgG1 Fc fusion proteins (see Section 5.6,above) for monoclonal antibody selection and purification. Specifically,five male xeonmice mice were immunized with the fusion protein accordingto standard protocols and spleen cells from a mouse that showed positivereactivity to the 103 fusion protein were fused with SP2/0 myeloma cellsusing standard polyethylene glycol (PEG) techniques (see, e.g., Harlowand Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Current Protocols inImmunology, 1992, Coligan, J. E. et al., Eds., John Wiley & Sons, NY).

The resulting hybridoma cell lines were screened to select clones whichsecreted antibodies that bound specifically to the human 103 geneproduct by ELISA and Biacore (BIAcore, Inc.; Uppsala, Sweden) asdescribed in Fagerstam et al., 1992, J. of Chromatography 597:397-410and in Kretschmann & Raether, 1968, Z. Naturforschung Teil. A. 23:2135.These assays used the above-described plate bound human 103-mouse IgG1Fc fusion protein and control fusion proteins containing the same murineIgG1 Fc fusion to select monoclonal antibodies that specifically boundto a human 103 gene product but did not bind to a control Fc fusionprotein. Selected cell lines were cloned using ClonalCell™-HY Medium D(StemCell Technologies, Inc., Vancouver, BC) according to themanufacturer's recommendations. Supernatants from the monoclonal celllines were screened for binding to cells presenting the human 103 geneproduct on their surface.

6.8.2. RESULTS

Anti-human 103 monoclonal antibodies were generated, as described inSection 6.8.1, above, in xenomice expressing human IgG2. Thus, theantibodies generated by spleen cells isolated from these mice were, infact, human antibodies against a human 103 gene product.

Supernatants from the hybridoma cell line clones derived from thesespleenocytes were screened for binding to the human 103 gene productexpressed on the surface of 293T cells transfected with the third human103 fusion protein described in Section 6.8.1, above. Several clonesshowed significant binding to these 103 gene expressing cells,indicating binding of monoclonal antibodies to the human 103 geneproduct antigen expressed on the cell surface. Two clones in particular,referred to herein as MA6 4C7.2 and MA6 10N13.3, showed very strongstaining.

Five clones, including the MA6 4C7.2 and MA6 10N13.3 clones describedhereinabove, were purified from DMEM 3% FCS (Ultra-low IgG, Gibco BRL)supplemented with Penicillin/Streptomycin and L-glutamine, and themonoclonal antibodies produced by these clones were purified by ProteinA affinity chromatography. The binding affinities of these antibodiesfor the human 103 gene product antigen were evaluated by Biacore(BIAcore, Inc.; Uppsala, Sweden) as described in Fagerstam et al., 1992,J. of Chromatography 597:397-410 and in Kretschmann & Raether, 1968, Z.Naturforschung Teil. A. 23:2135. The K_(d) values thus determined rangedfrom 1.5×10⁻⁷ to 4×10⁻⁹ M.

6.9. IDENTIFICATION OF A 103 GENE PRODUCT LIGAND

This example presents data demonstrating the existence of a previouslyunknown 103 gene product ligand expressed by mice spleen cells.Specifically, the example describes an assay wherein binding of a 103gene product to mouse spleen cells is detected. Data is also presentedwherein monoclonal antibodies to the human 103 gene product areidentified and shown to specifically block binding of the 103 geneproduct to this novel ligand.

6.9.1. MATERIALS AND METHODS

103 Fusion Proteins:

Fc fusion proteins of the 103 gene product were prepared as described inSection 6.6.1, above.

Monoclonal Antibodies:

Monoclonal antibodies that specifically bind to the extracellular domainof a human 103 gene product were prepared as described in Section 6.6.1,above.

Immunization and Preparation of Tissue Sections:

Approximately 4 month old C57BI/6 mice were immunized subcutaneously atmultiple sites along the back, intraperitoneally, and at the base of thetail with a total volume of approximately 200 μl of a 1:1 emulsion ofFreunds complete adjuvant (Sigma, St. Louis, Mich.) and phosphatebuffered saline. Spleens were harvested from unimmunized mice and fromimmunized mice 1, 3, 5 and 14 days after immunization. The harvestedspleens were frozen in OCT 4583 embedding medium (Tissue-Tek). Freshfrozen 8 μm sections were prepared, fixed in acetone for 10 minutes, andwashed in phosphate buffered saline (PBS) for five minutes. Sectionswere then blocked for 30 minutes in PBS containing 5% goat serum and 1mg/ml mIgG (Rockland, Inc.).

103 Binding Assays:

Tissue sections of spleen cells from unimmunized and immunized mice,prepared as described above, were incubated with human 103 geneproduct-Fc fusion protein or with human IgG1 (10 μg/ml in PBS/1% goatserum) for one hour at room temperature and then washed three times inPBS. Bound Fc-fusion proteins were detected by incubating the sectionswith alkaline phosphatase-conjugated anti-human IgG1 antibodies (10μg/ml in PBS/1% goat serum; Jackson Laboratories) for 30 minutes.Following these washes in PBS alkaline phosphatase, activity wasvisualized with a BCIP/NBT(5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium) substratekit from Vector Laboratories.

Monoclonal antibodies to the human 103 gene-Fc fusion protein weretested for their ability to block binding of the 103 gene-Fc fusionprotein to spleen sections by adding a 60 to 100 fold molar excess ofthe purified monoclonal antibodies to 10 μg/ml 103 gene-Fc fusionprotein in PBS/1° A goat serum and incubating the tissue sections withthe mixture as described above.

6.9.2. RESULTS

Spleen sections from unimmunized mice and immunized mice were screenedwith purified human 103 gene-Fc fusion protein, as described in Section6.8.1, above, for binding sites that specifically bind to the 103 geneproduct. Immunization dependent binding of the fusion protein wasobserved to scattered cells in the red pulp of spleens. However, nobinding of hIgG1, which was used as a control protein, was observed toany of the spleens analyzed. These binding experiments were repeatedwith two other groups of mice and were found to be repeatable. Theresults were further confirmed using murine 103-human IgG1-Fc fusionprotein and with murine 103-alkaline phosphatase fusion proteins(prepared as described in Section 6.3.1, above). Immunization dependentbinding of the murine 103-alkaline phosphatase fusion protein toscattered cells in the T cell zones of the white pulp of the spleens wasfurther detected.

Binding of the murine 103 gene-alkaline phosphatase fusion protein tospleen cells was blocked by incubating in the presence of a 100 foldexcess of murine 103 gene-human IgG1-Fc fusion protein. However, bindingof the murine 103-gene alkaline phosphatase fusion protein was notblocked when incubated in the presence of control murine IgG1(anti-TNP). These results demonstrate that immunization leads to theregulation of binding sites for the 103 gene product on a subpopulationof cells in the spleen and/or the migration of cells in the spleen whichhave binding sites to the 103 gene product.

To further characterize the above-described binding of the 103 geneproduct to its ligand in spleen cells, five anti-human 103 monoclonalantibodies, prepared as described in Section 6.6.1, above, were testedfor their ability to block this binding. The monoclonal antibodiestested included the monoclonal antibodies M15 1B4.1, M15 A16.1, M159F11.1, and M15 3F7.3, which are described in Section 6.6.2, above, andalso the monoclonal antibody produced by the hybridoma cell linereferred to as M15 10F7.1, which produces a monoclonal antibody of theisotype IgG2a Kappa. Incubation with a 100 fold excess of M15 10F7.1,M15 1B4.1 or M15 5A16.1 monoclonal antibody completely abolished bindingof the 103 gene-Fc fusion protein to spleens cells from immunized miceprepared 3, 5 or 14 days post immunization. Incubation with a 70 foldexcess of M15 9F11.1 or with a 60 fold excess of M15 3F7.3 monoclonalantibody only partially blocked the 103 gene-Fc fusion protein bindingto these spleen cells. As controls, the 103 gene-Fc fusion protein wasalso incubated with two irrelevant isotype-matched monoclonalantibodies: anti-TNP, an IgG2a isotype; and anti-hT075, an IgG1 isotype.Incubation with these control antibodies did not block binding of the103 gene-Fc fusion protein to spleen cells. Thus, binding of the 103gene product to its ligand in spleen cells can be effectively blocked,e.g., by administering antibodies that compete with the 103 gene productfor its ligand.

7. MICROORGANISM DEPOSITS AND REFERENCES CITED

The murine hybridomas listed below were deposited with the American TypeCulture Collection (ATCC™), 10801 University Blvd., Manassas, Va. 20110,under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure, and compliance with the criteria set forth in 37 C.F.R.§1.801-1.809 regarding availability and permanency of deposits. Themurine hybridomas were produced as described above in Section 6.6.1. Thedeposits were made on the date indicated and assigned the indicatedaccession number:

Microorganism Deposit ATCC ™ No. Date of Deposit M15 3F7.3 PTA-593 Aug.24, 1999 M15 9F11.1 PTA-590 Aug. 24, 1999 M15 2O3.1 PTA-591 Aug. 24,1999 M15 5A16.1 PTA-587 Aug. 24, 1999 M15 10F7.1 PTA-592 Aug. 24, 1999M15 1B4.1 PTA-588 Aug. 24, 1999

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety and for all purposesto the same extent as if each individual publication or patent or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

What is claimed is:
 1. A method for treating asthma in a mammaliansubject, comprising administering to the subject a pharmaceuticalcomposition comprising a monoclonal antibody produced by: (a) hybridomaclone M15 3F7.3 (ATCC™ No. PTA-593); (b) hybridoma clone M15 203.1(ATCC™ No. PTA-591); (c) hybridoma clone M15 10F7.1 (ATCC™ No. PTA-592);(d) hybridoma clone M15 1B4.1 (ATCC™ No. PTA-588); (e) hybridoma cloneM15 9F11.1 (ATCC™ No. PTA-590); or (f) hybridoma clone M15 5A16.1 (ATCC™No. PTA-587); or an antigen binding fragment thereof, and apharmaceutically acceptable carrier.
 2. A method for treating allergy ina mammalian subject, comprising administering to the subject apharmaceutical composition comprising a monoclonal antibody produced by:(a) hybridoma clone M15 3F7.3 (ATCC™ No. PTA-593); (b) hybridoma cloneM15 203.1 (ATCC™ No. PTA-591); (c) hybridoma clone M15 10F7.1 (ATCC™ No.PTA-592); (d) hybridoma clone M15 1B4.1 (ATCC™ No. PTA-588); (e)hybridoma clone M15 9F11.1 (ATCC™ No. PTA-590); or (f) hybridoma cloneM15 5A16.1 (ATCC™ No. PTA-587); or an antigen binding fragment thereof,and a pharmaceutically acceptable carrier.
 3. A method for treating anIgE-mediated immune disorder in a mammalian subject, comprisingadministering to the subject a pharmaceutical composition comprising amonoclonal antibody produced by: (a) hybridoma clone M15 3F7.3 (ATCC™No. PTA-593); (b) hybridoma clone M15 203.1 (ATCC™ No. PTA-591); (c)hybridoma clone M15 10F7.1 (ATCC™ No. PTA-592); (d) hybridoma clone M151B4.1 (ATCC™ No. PTA-588); (e) hybridoma clone M15 9F11.1 (ATCC™ No.PTA-590); or (f) hybridoma clone M15 5A16.1 (ATCC™ No. PTA-587); or anantigen binding fragment thereof, and a pharmaceutically acceptablecarrier.